We studied the effects of caffeine on calcium transport by subcellular organelles isolated from rabbit myocardium. Caffeine increased myofibrillar basic and calcium-activated ATPase activity at 20 mil but not at lower concentrations. Mitochondrial and sarcoplasmic reticulum (SR) calcium accumulation was measured both by dual wavelength spectrophotometry with the calcium-sensitive dye, murexide, and by Millipore filtration with 45Ca. In mitochondria, caffeine impaired phosphate-assisted calcium transport but did not alter the closely related parameters of oxygen uptake, P/O ratio (nmol adenosine diphosphate consumed/n ats oxygen consumed, state 3 respiration) or limited calcium loading. In SR, caffeine impaired calcium accumulation. New methods were used to characterize calcium accumulation in the absence of oxalate according to first order reaction kinetics. Caffeine increased the rate constant while decreasing calcium accumulated. It also increased associated calcium-activated ATPase activity at low (30 μIM) but not high (240 μJM) external calcium concentration. In the presence of oxalate, caffeine decreased the rate of calcium accumulation, more with low than high calcium concentration. Net efflux of 45Ca from preloaded SR also was increased by caffeine. The findings indicate that caffeine impairs active calcium accumulation by making SR vesicle membranes more permeable to calcium.