Adenoviral vectors have been successfully used to increase the activity of the sarcoplasmic reticulum Ca(2+)-ATPase in adult ventricular myocytes and to produce functional improvements in contractility in vivo and in vitro. While in vivo experiments are often performed in rat, in vitro manipulation of myocytes has been confined to rabbit and human cells. In the present study we make quantitative comparisons between cultured adult rat and rabbit myocytes in their responses to SERCA2a overexpression using adenoviral vectors. We also compare the strategy of SERCA2a overexpression with that of phospholamban down-regulation, using adenovirus carrying antisense message, as a means to increase SERCA2a activity and enhance contraction and relaxation. Adult myocytes were cultured for 48 h with either vector, and contraction assessed in 2 mM Ca2+, 37 degrees C, at a range of stimulation frequencies. Contraction amplitude was enhanced to a similar degree in either rat or rabbit myocytes at most stimulation frequencies, with SERCA2a overexpression and phospholamban down-regulation approximately equally effective. The maximum effect of either vector was less than that of beta-adrenoceptor agonists. Relaxation was accelerated in rabbit myocytes more strongly than in rat. Phospholamban antisense was slightly less effective than SERCA2a overexpression on relaxation times in rabbit. Increasing stimulation frequency also accelerated relaxation in rat myocytes: this effect was greater than, and additive with, that of SERCA2a overexpression. We conclude that, despite some species-dependent modification, the effects of increased SERCA2a activity are broadly similar in rat and rabbit. Both SERCA2a overexpression and phospholamban down-regulation are effective strategies, and neither appears to produce supraphysiological stimulatory effects on contraction or relaxation.