The calmodulin binding proteins, caldesmon and calponin, are cleaved by both major isoforms of calpain in vitro. The patterns of fragments generated by each enzyme are essentially identical for a given substrate. Qualitatively, the cleavage pattern of each substrate is unchanged by the presence or absence of calmodulin suggesting that the interaction between calmodulin and these calmodulin-binding proteins does not alter substrate recognition by calpain. However, calmodulin (at microM concentrations) does have a small, but significant, inhibitory effect directly on calpain as evidenced by slower rates of cleavage of alpha-casein, a protein that does not bind calmodulin. Inhibition is more pronounced with mu-calpain (15-25%) than with m-calpain (6-10%). In order to demonstrate, unequivocally, that substrate recognition does not require an interaction between calpain and a substrate's calmodulin-binding domain, recombinant, full-length caldesmon and a mutant lacking the calmodulin binding domain were tested as substrates for calpain in the presence and absence of calmodulin. Calpain produced similar cleavage patterns of the baculovirus expressed caldesmon and the truncated mutant. Competition experiments demonstrated that calpain does not discriminate between the truncated mutant and full length caldesmon. This suggests that substrate recognition by calpain was not altered significantly by the absence of the calmodulin-binding domain. Cleavage of a second calmodulin-binding protein, calponin was also examined. The rate of calponin cleavage was increased in the presence of calmodulin, an observation that is also inconsistent with any requirement for calpain to bind to its calmodulin-binding site. These results demonstrate that calmodulin-binding domains do not provide substrate recognition sites for calpains. It seems likely that the calmodulin-like regions of calpain function to bind calcium and to regulate enzyme conformation as required for activity and that they do not interact directly with most substrates.