Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35 nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation.