Villin, a Ca2+‐regulated actin‐binding protein is a major component of microvilli of intestinal epithelial cells and kidney proximal tubule cells. Villin expression during assembly of the brush border can be investigated using a human colon adenocarcinoma cell line HT29‐18. This cell line is able to differentiate under nutritional control and develops an enterocyte‐like phenotype. A cDNA library from a subclone HT29‐18‐C1 was constructed in an expression vector and a cDNA specific for human villin was isolated. This cDNA codes for the 110 carboxy‐terminal residues of villin. Within that region, the 76 carboxy‐terminal residues present 65% homology with the chicken villin ‘head piece’. We show that two mRNA species 4.0 kb and 3.2 kb long hybridize with this cDNA probe in humans, whereas in rat and chicken only one mRNA species can be detected. The two villin mRNA species are co‐expressed in normal human small and large intestinal mucosa and tumoral HT29‐18 cells as well as in normal kidney. No villin mRNAs were detected in other normal or malignant epithelial cell types. Finally, we observed an accumulation of the two mRNA species coding for villin when HT29‐18 cells become differentiated, suggesting that control of villin expression during terminal differentiation can occur at the transcription level or by RNA stabilization.