Background: Experiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre. Methods and results: Two lines of smooth muscle protein 22 alpha-Cre (SM22 alpha-Cre) mice were bred to foxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22 alpha-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22 alpha-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22 alpha-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry. Conclusions: SM22 alpha-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22 alpha-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22 alpha-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes. (C) 2012 Elsevier Inc. All rights reserved.