Caldesmon (CaD) has been suggested to regulate SM-contraction independently from MLC₂₀ phosphorylation. The whole protein and a 20kDa C-terminal actin binding fragment inhibit tension in skinned SM fibers. The function of the myosin binding domain, encoded by exon2, is not clear. To investigate the in vivo function of this domain, we generated a mouse line with a deletion of exon2 (ex2-/-), leading to an expression of a truncated CaD without myosin binding domain. The ablation was confirmed by southern blot analysis, cDNA-sequencing and peptide mass fingerprint. The ex2-/- mice are viable and produce fertile offsprings. Expression of the truncated CaD has no effect on the dose response relation to carbachol (EC₅₀ wt: 0.39± 0.14mM; ex2-/-: 0.33± 0.09mM) nor on the Ca ²⁺ sensitivity of contraction (pCa₅₀ wt: 6.49± 0.3; exon2-/-: 6.79± 0.03) in intact, and respectively, triton skinned longitudinal ileal SM. The lack of effect on force development was not due to a compensatory change in the expression of caldesmon, myosin, tropomyosin, calmodulin and MLCK. Moreover, no differences in isoform expression of MLC₁₇ and MHCs have been detected. These data imply that the myosin binding site of CaD is not essential for the regulation of SM contraction but might be involved in regulation of SM relaxation. (This work was supported by the DFG.)