We have investigated endothelin (ET)-regulated secretion of atrial natriuretic factor (ANF) from primary neonatal rat ventricular myocytes, where hormone release is thought to be constitutive. In a dose-dependent, nifedipine-sensitive manner, ET acutely enhanced ANF release by two- to fivefold over control cultures within 15 min of agonist exposure, demonstrating that ventricular myocytes display a primary characteristic of a regulated secretory cell type. Unlike atrial cultures, ET enhanced ANF release during the first 30 min of exposure; thereafter, secretion rates returned to control levels. KCl, however, effectively enhanced ANF release only during the first 15 min of exposure. Subcellular fractionation of ventricular culture homogenates did not reveal atrial-type dense secretory granules, and pulse-chase labeling experiments showed that the transit time of newly synthesized ANF was short in ventricular myocytes [time required for half of labeled ANF to be released from cells (t1/2) = 0.5-1.5 h) compared with atrial myocytes (t1/2 = 4 h). These results suggest that, whereas ventricular myocytes possess some of the characteristics of a constitutively secreting cell type (e.g., few, if any, dense secretory granules and rapid transit time for newly synthesized hormone); however, they also display the capacity for regulated secretion of ANF in response to the physiological agonist ET.