We evaluated various constructs to obtain cell specific expression of exogenous Ca²⁺ ATPase (SERCA) gene in cardiac myocytes, following cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous Enhanced Green Fluorescence Protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. While the CMV promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre dependent expression under control of the CMV promoter, and Cre expression under control of a cardiac specific promoter, yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immuno-staining of exogenous SERCA1 and endogenous SERCA2, and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell specific SERCA expression and a definite increase over endogenous Ca²⁺ ATPase activity, as well as faster removal of cytosolic calcium following membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell specific expression of exogenous SERCA is wanted in cardiac myocytes, following cDNA delivery to mixed cell populations.