Calcium binding to cultured whole cell monolayers, to their membranes made by "gas dissection," and to sarcolemmal vesicles from canine heart, is measured as a function of pH from 5.5-8.5. The effects on binding are compared to the contraction response of the cultured cells over the same pH range. All responses to pH, including calcium binding and contraction, are well described (r = 0.98-0.99) by a relation in which calcium binding depends primarily upon the extent of ionization of sarcolemmal binding sites. The effect of pH on calcium binding to phospholipid extracted from sarcolemmal vesicles suggests that the phospholipid accounts for at least 75% of the binding. The extent of ionization of the sites is pH-dependent and predicted by a form of the Henderson-Hasselbach relation with pK of the putative sites between 6.60 and 7.15. As pH increases from 5.5-8.5, membranous calcium binding, cellular calcium uptake, and contraction amplitude increase proportionally. Cellular calcium uptake increases by 4.4 mmol/kg dry weight cells (0.75 mmol/kg wet weight). Sixty percent to 65% of this increment is rapidly exchangeable and lanthanum displaceable, indicative of sarcolemmal localization. The remainder enters a slowly exchangeable (t1/2 = 26 minutes) compartment not directly related to support of contraction. The study supports the hypothesis that calcium bound to sarcolemmal sites plays a critical role in control of myocardial contraction.(ABSTRACT TRUNCATED AT 250 WORDS)