The actin-binding protein calponin has been implicated in the regulation of smooth muscle contraction. We have isolated cDNA clones encoding a novel acidic calponin isoform from rat aortic vascular smooth muscle cells. The initial 273 residues of the deduced 330 amino acid polypeptide (M(r) 36,377) are highly homologous to basic smooth muscle calponin isoforms, but the remaining 57 residues at the carboxyl terminus comprise a unique and strongly acidic domain. The sequence of the acidic domain shows high homology (93.3% identity) to the partial sequence of HUMXT01244, an unidentified human hippocampal gene product (Adams, M., Dubnick, M., Kerlavgne, A. R., Moreno, R., Kelly, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992) Nature 355, 632-634). Transcripts encoding acidic calponin are expressed in cultured rat aortic vascular smooth muscle cells and in non-muscle and smooth muscle tissues of adult rat. Based on its calculated M(r) and the tissue distribution of its expression, acidic calponin is an excellent candidate for a previously detected non-muscle calponin homolog (Takeuchi, K., Takahashi, K., Abe, M., Nishida, W., Hiwada, K., Nabeya, T., and Maruyama, K. (1991) J. Biochem. (Tokyo) 109, 311-316). Like basic calponin isoforms, acidic calponin synthesized in a bacterial expression system bound F-actin. However, unlike basic calponin, the acidic isoform did not interact with Ca2+/calmodulin, indicating a functional distinction between the muscle and non-muscle forms.