Targeted gene deletion in mice provides a powerful approach to determining the physiological roles of μ- and m-calpain and the opportunity to approach their isoform specific functions. Initial studies targeted Capn4 based on the prediction that loss of this calpain subunit would abolish activity of both μ- and m-calpain. Capn4-/- murine embryos died between days 10 and 11 of gestation, and there was no detectable μ- or m-calpain activity in these or younger embryos [31]. Capn4-/- murine embryonic fibroblasts (MEFs) could be cultured from these embryos, although they also lacked calpain activity as assessed by casein zymography or by the formation of characteristic spectrin breakdown products, and they displayed migration defects consistent with a role for calpain in release of focal adhesions [9]. An independently derived Capn4 knockout, involving a more extensive deletion of the gene, resulted in an earlier embryonic lethality, apparently at a pre-implantation stage [32]. The different times of embryonic lethality suggested that the first reported Capn4-/- mice [31] were targeted with a hypomorphic mutation, which retained some small level of calpain activity, allowing for their survival to mid-gestation, while the second reported Capn4-/- mice [32] represented a true null mutation. Disruption of Capn1, encoding the μ-calpain catalytic subunit, was subsequently reported to result in fertile, viable mice with some mild defects in the μ-calpain rich platelets relating to their aggregation and clot retraction [33]. The fact that Capn4 null mice die during embryogenesis indicates that at least one of the ubiquitous calpains is essential for development to term. The viability of Capn1-deficient mice does not however distinguish between two possibilities: either that m-calpain is specifically required during embryogenesis, or that either form of calpain alone is sufficient and can compensate for the absence of the other. To resolve this question, we have now knocked out the Capn2 gene encoding the m-80 k subunit in mice. We report here that Capn2 null embryos died prior to the implantation stage, indicating that m-calpain is indispensable for early embryogenesis. This role cannot be fulfilled by μ-calpain, which is expressed in embryonic stem (ES) cells [31] and is assumed to be present at this stage of gestation. This demonstrates unequivocally that m-calpain and μ-calpain have distinct physiological roles during early embryogenesis.