Tissue-specific knockouts generated through Cre-loxP recombination have become an important tool to manipulate the mouse genome. Normally, two successive rounds of breeding are performed to generate mice carrying two floxed target-gene alleles and a transgene expressing Cre-recombinase tissue-specifically. We show herein that two promoters commonly used to generate endothelial-specific (Tie2) and smooth muscle-specific (smooth muscle myosin heavy chain, Smmhc) knockout mice, exhibit activity in the female and male germ lines, respectively. This can result in the inheritance of a null allele in the second generation which is not tissue-specific. Careful experimental design is required therefore to ensure that tissue-specific knockouts are indeed tissue-specific and that appropriate controls are used to compare strains.