Orlistat is an anti-obesity treatment with which several gastrointestinal (GI) side-effects are commonly associated in the initial stages of therapy. There is no physiological explanation as to why two-thirds of those who take the drug experience one or more side-effects. It has been hypothesized that the GI microbiota may protect from or contribute to these GI disturbances. Using in vitro batch culture and human gut model systems, studies were conducted to determine whether increased availability of dietary lipids and/or orlistat affect the composition and/or activity of the faecal microbiota. Results from 24-h batch culture fermentation experiments demonstrated no effect of orlistat in the presence or absence of a dietary lipid (olive oil) on the composition of bacterial communities [as determined by fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses], but did show there was great variability in the lipolytic activities of the microbiotas of individuals, as determined by gas chromatography analysis of long-chain fatty acids in samples. Subsequent studies focused on the effect of orlistat in the presence and absence of lipid in in vitro human gut model systems. Systems were run for 14 days with gut model medium (GMM) only (to steady state, SS), then fed at 12-h intervals with 50 mg orlistat, 2 g olive oil or a mixture of both for 14 days. FISH and DGGE were used to monitor changes in bacterial populations. Bacteria were cultivated from the GMM only (control) systems at SS. All strains isolated were screened for lipolytic activity using tributyrin agar. FISH and DGGE demonstrated that none of the compounds (singly or in combination) added to the systems had any notable effect on microbial population dynamics for any of the donors, although Subdoligranulum populations appeared to be inhibited by orlistat in the presence or absence of lipid. Orlistat had little or no effect on the metabolism of indigenous and added lipids in the fermentation systems, but there was great variability in the way the faecal microbiotas of the donors were able to degrade added lipids. Variability in lipid degradation could be correlated with the number and activity of isolated lipolytic bacteria. The mechanism by which orlistat and the GI microbiota cause side-effects in individuals is unknown, but several hypotheses have been proposed to account for their manifestation. The demonstration of great variability in the lipolytic activity of microbiotas to degrade lipids led to a large-scale cultivation-based study of lipolytic/lipase-positive bacteria present in the human faecal microbiota. Of 4,000 colonies isolated from 15 donors using five different agars, 378 strains were identified that had lipase activity. Molecular identification of strains isolated from five donors demonstrated that lipase activity is more prevalent in the human GI microbiota than previously thought, with members of the phyla Firmicutes, Bacteroidetes and Actinobacteria identified. Molecular identification and characterization of the substrate specificities of the strains will be carried out as part of ongoing work.