Cells digested from adult rat heart and cultured for 14 days demonstrate all the structural elements, in mature form, associated with the process of excitation-contraction (EC) coupling. The transverase tubular (TT) system is well developed with an extensive junctional sarcoplasmic reticulum (JSR). In nonphosphate-containing buffer contraction of the cells is lost as rapidly as zero extracellular Ca concentration ([Ca]o) solution is applied (less than 10 s) and a negative contraction staircase is produced on increase of stimulation frequency. Structurally and functionally the cells have the characteristics of adult cells in situ. 45Ca exchange and total 45Ca measurement in N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES)-buffered perfusate define three components of cellular Ca: a rapidly exchangeable component (t1/2 less than 25 s) accounting for 36% of total Ca, a slowly exchangeable component (t1/2 53 min) accounting for 7% of total Ca, and the remaining 57% cellular Ca is "inexchangeable" (demonstrates no significant exchange within 60 min). The slowly exchangeable component can be increased 10-fold within 60 min by addition of phosphate to the perfusate. The Ca distribution and exchange characteristics are little different from those of 3-day cultures of neonatal rat heart previously studied [Langer, G. A., J. S. Frank, and L. M. Nudd. Am. J. Physiol. 237 (Heart Circ. Physiol. 6): H239-H246, 1979]. The results suggest that the cells are representative of adult cells in situ and that both sarcolemmal-bound and sarcoplasmic reticular Ca contribute to the component of Ca that is rapidly exchangeable.