The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.