Minces of several organs from the transgenic mouse ROSAβ-gal 26 (ROSA-26), which robustly expresses bacterial lac-Z in most tissues, were exposed to 4-bromo, 5-chloro, 3-indoyl, β-D-galactopyrosanide (X-gal) at pH ranging from 7.5 to 9.5 to determine the optimal pH for in situ demonstration of bacterial β-galactosidase activity (neutral pH optimum) while minimizing detection of potentially confounding endogenous mammalian β-galactosidase (acidic pH optimum). Similar studies were performed with organ minces from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and macaques to confirm the effect of pH on minimizing detection of endogenous mammalian β-galactosidase. In all organs evaluated; heart, liver, spleen, kidney, brain, and skeletal muscle, endogenous β-galactosidase activity was rarely detected following incubation at pH greater than 7.5. In contrast, bacterial β-galactosidase activity in the ROSA-26 mice was strongly detected in organ minces following incubation at pH 8.0–9.0. These findings are similar to previous observations we have made in lung minces and confirm that a simple alteration of a commonly used histochemical technique for detecting in situ β-galactosidase activity, raising the reaction buffer pH to weakly alkaline range, can reliably distinguish between endogenous activity and that resulting from exogenous bacterial gene expression.