To generate temporally controlled targeted somatic mutations selectively and efficiently in skeletal muscles, we established a transgenic HSA‐Cre‐ER^T2 mouse line in which the expression of the tamoxifen‐dependent Cre‐ER^T2 recombinase is under the control of a large genomic DNA segment of the human skeletal muscle α‐actin gene, contained in a P1‐derived artificial chromosome. In this transgenic line Cre‐ER^T2 is selectively expressed in skeletal muscles, and Cre‐ER^T2‐mediated alteration of LoxP flanked (floxed) target genes is skeletal muscle‐specific and strictly tamoxifen‐dependent. HSA‐Cre‐ER^T2 mice should be of great value to analyze gene function in skeletal muscles, and to establish animal models of human skeletal muscle disorders. genesis 41:165–170, 2005. © 2005 Wiley‐Liss, Inc.