This preliminary study reports for the first time that there might be a possible association between bacteria and the aetiology of some cholesterol calculi. The gall-bladder biles from 225 cholecystectomy patients underwent bacteriological and microscopic study. Cholesterol calculi from 13 patients (10.2%) were observed to be associated with gall-bladder bile profusely infected with at least one bacterial species that was shown to possess beta-glucuronidase activity, an enzyme that is thought to promote calcium bilirubinate precipitation in bile. Concomitantly, the associated gall-bladder bile was 'high' in calcium bilirubinate precipitation and the precipitate was also detected in the centre of the gallstones. Moreover, in approximately half of these patients (six of 13), the cholesterol gallstones' nuclear areas also contained calcium palmitate, which is also thought to be due to bacterial activity. The results also support the hypothesis that bacteria with active beta-glucuronidase (for example, Escherichia coli) can significantly influence the aetiology of brown pigment gallstones. In contrast, bacteria were observed to have no role in black pigment gallstone formation, as their associated gall-bladder biles were always observed to be sterile.