In vivo, butyrate is a major energy source for the colonic epithelium and is thought to stimulate proliferation. In contrast, butyrate in vitro has been shown to inhibit proliferation and induce differentiation and apoptosis in colonic epithelial cells. Most colon cell cultures are grown in medium containing high concentrations of glucose, whereas in vivo, the main energy source used by the colon cells is butyrate. The aim of this study was to determine whether the apparent contrasting roles of butyrate in vivo and in vitro could be as a consequence of differences in glucose availability. The sensitivity of two human colorectal tumour cell lines, one adenoma (S/RG/C2) and one carcinoma (HT29) to butyrate-induced growth inhibition and apoptosis was investigated to determine whether these cellular effects were altered under glucose depleted culture conditions. Glucose depletion resulted in increased apoptosis in both cell lines in the absence of butyrate. Butyrate in standard culture conditions (containing 25 mM glucose and 1 mM pyruvate) inhibited growth and induced apoptosis in both cell lines. However, low concentrations of butyrate in glucose depleted culture conditions (i.e. standard growth medium without glucose and pyruvate supplements) were found to reduce apoptosis induced by glucose deprivation and increase cell yield in both cell lines. The results show that in glucose depleted culture conditions, butyrate at low concentrations (0.5 mM for S/RG/C2, and 0.5 and 2 mM for HT29 cells) was found to be growth stimulatory whereas in the presence of glucose, these same concentrations of butyrate induced apoptosis. Thus, whether butyrate is growth stimulatory or growth inhibitory may depend on the availability of other energy sources. These observations may, in part, provide an explanation for the apparent opposite effects of butyrate on proliferation reported in vivo and in vitro.