The membrane location and mechanism of inositol 1,3,4,5-tetrakisphosphate (InsP4)-regulated Ca2+ uptake in cardiac membrane vesicles was investigated. In canine and rat membranes separated by sucrose density gradient centrifugation, InsP4-regulated Ca2+ uptake was slightly more enriched in low density than in higher density membranes. Membranes supporting InsP4-regulated Ca2+ uptake were correspondingly enriched in type 1 InsP3 receptors. Junctional sarcoplasmic reticulum (J-SR), enriched in sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and ryanodine receptors, separated predominantly with higher density membranes. In membranes supporting InsP4-regulated Ca2+ uptake, Ca2+ uptake was facilitated by a high Ca2+ affinity carrier that was insensitive to thapsigargin. Ca2+ uptake in J-SR was mediated by thapsigargin-sensitive SERCA2a. Net Ca accumulation was enhanced by oxalate in both SR subtypes. Although Ca2+-carrier-mediated Ca2+ uptake was ATP independent, ATP indirectly regulated net Ca2+ accumulation by modifying Ca2+ efflux via a Ca2+ channel with properties of type 1 InsP3 receptors. In the presence of < or = 0.1 mM ATP, InsP4 enhanced Ca2+ accumulation whereas InsP4 inhibited Ca2+ uptake at higher ATP concentrations. In the presence of 0.15 mM ATP, InsP4 stimulated Ca2+ efflux from vesicles preloaded with Ca. Several other InsP4 isomers and 1,3,4-InsP3 also stimulated Ca2+ efflux but with slightly less potency than 1,3,4,5-InsP4. Ruthenium red enhanced net Ca accumulation by the Ca2+ carrier and reduced the potency of ATP, InsP4, and InsP3 to stimulate Ca2+ efflux in vesicles. In summary, this investigation shows that a Ca2+ carrier facilitates Ca loading in a sarcoplasmic reticulum subtype distinct from J-SR. InsP4 and InsP3 are proposed to regulate Ca2+ efflux in low density SR by acting on an ATP-modulated Ca2+ channel with properties of type 1 InsP3 receptors.