We investigated the effects of the basic peptides polyarginine, protamine, and polylysine on dispersed bovine parathyroid cells. All three peptides produced a dose-dependent inhibition of dopamine-stimulated cAMP accumulation, with half-maximal inhibition at 4 x 10(-8), 1.5 x 10(-7), 3 x 10(-7), and 2 x 10(-6) M, respectively, for polyarginine, protamine, and two preparations of polylysine of molecular weights 10,200 and 3800. The inhibition of cAMP accumulation was reversible and was blocked by preincubating the cells overnight with 0.5 micrograms/ml of pertussis toxin. The same peptides also inhibited PTH release at similar concentrations, markedly stimulated the accumulation of inositol phosphates at two- to threefold higher concentrations, and produced transient increases in the cytosolic Ca2+ concentration (Cai) in fura-2-loaded parathyroid cells. The polylysine-evoked spike in Cai persisted despite the removal of extracellular Ca2+, indicating that it arose from intracellular Ca2+ stores. Exposure of the cells to elevated extracellular magnesium (Mg2+) concentrations elicited a similar spike in Cai but blocked the Cai transient in response to subsequent addition of polylysine, or vice versa. Thus, Mg2+ and polylysine mobilize Ca2+ from the same intracellular store(s). These results indicate that highly basic peptides closely mimic the effects of polyvalent cations on parathyroid function, suggesting that both agents may regulate parathyroid function via similar biochemical pathways.