Histochemical and immunocytochemical methods were used to study the presence of carbonic anhydrase (CA) isoenzymes in the caecum and colon of normal and germ-free rats. Very high enzyme activity was demonstrated by histochemistry in the caecum and proximal colon of normal rats, while the activity decreased in the distal colon. Very strong immunostaining for the isoenzyme CA I was found in the cytoplasm of surface cells and upper gland cells in the caecum and colon of normal rats. In the distal colon the staining was less intense with a marked cell-to-cell variation. Ca II was found in the apical (luminal) cell region of the surface epithelium in all regions. Ca III was possibly present in small amounts, but this could not be judged with certainty. There was no difference in carbonic anhydrase between normal and germ-free rats (except for less staining of the mucosal capillaries in germ-free animals). Therefore, our data give no support to the hypothesis that CA I participates in the absorption of microbial fermentation products. The location of CA II in the apical cell region suggests a role for this isoenzyme in regulation of the microclimate close to the epithelial cells.