The potent collagenolytic activity of nonspecific proteinases suggested their use as enzymatic agents for the dissociation of single adult heart cells. This was assessed in guinea pig hearts perfused for 1 min with solutions containing hyaluronidase (100–10000 U/mL), trypsin (100–10000 U/mL), crude collagenase (100–500 U/mL), or nonspecific protease (0.1–100 U/mL). No rod-shaped cells were observed among the cells isolated with these concentrations of hyaluronidase, trypsin, or crude collagenase. By contrast, 45–80% of the cells released with nonspecific protease (5–10 U/mL) were rod shaped and Ca2+ tolerant. Resting and action potentials recorded from cells dispersed with nonspecific protease were similar to those recorded from cells isolated after prolonged collagenase exposure.