Ubiquitous μ‐ and m‐calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80‐kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28‐kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of μ‐ and m‐calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue‐specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the μ‐ and m‐calpain catalytic subunits, and had no detectable μ‐ and m‐calpain activities. These defects were corrected with a capn4‐encoding lentivirus. genesis 44:297–303, 2006. © 2006 Wiley‐Liss, Inc.