The duration of contraction in isolated electrically driven preparations from atrium and ventricle of mouse, rat, rabbit, guinea-pig and dog was consistently shorter in atrial compared to ventricular preparations. Overexpression of phospholamban (PLB) in transgenic mice prolonged duration of contraction, underscoring the importance of PLB for kinetics of cardiac contractility. The expression of regulatory proteins was studied by Western and Northern blot analysis. In rat myocardium, expression of the sarcoplasmic reticulum Ca²⁺ATPase (SERCA) was higher in atrium than in ventricle, as was also observed in the rabbit, guinea-pig and wild-type mouse samples. Canine myocardium, however, had similar levels of SERCA (protein and mRNA) in atrium and ventricle. PLB and calsequestrin on protein and RNA levels were lower in atrium than in ventricle from rat, rabbit, guinea-pig and wild-type mouse. PLB protein and RNA levels were higher in ventricle than in atrium at ages 1 and 5 days postnatally and in adult rats. SERCA protein and RNA levels were higher in ventricle than in atrium at days 1 and 5 after birth, but lower in ventricle than in atrium in adult rats. In dog, the calsequestrin level was identical in atrium and ventricle (protein and mRNA) and PLB did not differ between atrium vs ventricle at the protein level but was lower at the mRNA level. Also, Ca²⁺uptake was higher in atrium than in ventricle in the dog samples. The expression of the inhibitory subunit of troponin was unchanged between atrium and ventricle in all species studied (protein and mRNA). In dog, protein expression of triadin and junctin was lower in atrium vs ventricle. Triadin mRNA was not altered in dog atrium vs ventricle. In summary, while the hastened relaxation of atrium vs ventricle correlates in part with the lower expression of PLB and higher expression of SERCA, altered regional expression of other SR proteins handling Ca²⁺may also play an important role in some species.