In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F_2α(PGF_2α), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF_2α — and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF_2α treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF_2α increased the beat rate to 3.3 times that of control. After exposure to the β-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF_2α, 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF_2α by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF_2α. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal α-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal α-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.