Cre-loxP technology for conditional gene inactivation is a powerful tool in cardiovascular research. Induction of gene inactivation can be carried out by per oral or intraperitoneal tamoxifen administration. Unintended transient cardiomyopathy following tamoxifen administration for gene inactivation has recently been reported. We aimed to develop a protocol for tamoxifen-induced gene inactivation with minimal effects on gene transcription and in vivo cardiac function, allowing studies of acute loss of the targeted gene. In mRNA microarrays, 35% of the 34,760 examined genes were significantly regulated in MCM(+/0) compared with wild type. In MCM(+/0), we found a correlation between tamoxifen dose and degree of gene regulation. Comparing one and four intraperitoneal injections of 40 mg·kg(-1)·day(-1) tamoxifen, regulated genes were reduced to 1/5 in the single injection group. Pronounced alteration in protein abundance and acute cardiomyopathy were observed after the four-injection protocols but not the one-injection protocol. For verification of gene inactivation following one injection of tamoxifen, this protocol was applied to MCM(+/0)/Serca2(fl/fl). Serca2 mRNA levels and protein abundance followed the same pattern of decline with one and four tamoxifen injections. The presence of the MCM transgene induced major alterations of gene expression while administration of tamoxifen induced additional but less gene regulation. Thus nonfloxed MCM(+/0) should be considered as controls for mice that carry both a floxed gene of interest and the MCM transgene. One single tamoxifen injection administered to MCM(+/0)/Serca2(fl/fl) was sufficient for target gene inactivation, without acute cardiomyopathy, allowing acute studies subsequent to gene inactivation.