The steroid receptor coactivator-3 (SRC-3), also known as AIB1, ACTR, p/CIP and NCOA3, is a transcriptional coactivator for nuclear receptors and certain other transcription factors. SRC-3 is widely expressed and plays important physiological functions and pathogenic roles in breast and prostate cancers. SRC-3 knockout (SRC-3^-/-) mice display genetic background-dependent embryonic lethality and multiple local and systemic abnormalities. Since both the partial lethality and the systemic effects caused by global SRC-3 knockout interfere with downstream investigation of tissue-specific function of SRC-3, we have generated floxed SRC-3 (SRC-3^f/f) mice with conditional alleles carrying loxP sites in introns 10 and 12 by a gene-targeting strategy. The two SRC-3^f/f mouse lines (A and B) are indistinguishable from wild type mice. To test if deletion of the floxed exons 11 and 12 for SRC-3 nuclear receptor interaction domains and disruption of its downstream sequence for transcriptional activation domains would inactivate SRC-3 function, SRC-3^f/f mice were crossbred with EIIa-Cre mice to generate SRC-3^d/d mice with germ line deletion of the floxed SRC-3 gene. Both lines of SRC-3^d/d mice exhibited growth retardation and low IGF-I levels, which was similar to that observed in SRC-3^-/- mice. The line A SRC-3^d/d mice showed normal viability, while line B SRC-3^d/d mice showed partial lethality similar to SRC-3^-/- mice, probably due to variable distributions of genetic background during breeding. These results demonstrate that the floxed SRC-3 mouse lines have been successfully established. These mice will be useful for investigating the cell type- and developmental stage-specific functions of SRC-3.