Protamine sulphate (PS) effect on spontaneous and calcium-induced rhythmic contractions of isolated virgin rat uteri was studied. PS caused dose-dependent relaxation of both types of contractions (two-way ANOVA, significant dose effects). Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-5) mol/l), methylene blue (MB; 0.9 x 10(-6) mol/l) or propranolol (1.7 x 10(-5) mol/l) enhanced PS-mediated uterine muscle relaxation of spontaneous contractions. Dosedependent relaxation of spontaneous active isolated rat uterus with PS was lower in uteri pretreated with single dose of tetraethylammonium (TEA; 6 x 10(-3) mol/l), glibenclamide (2 x 10(-6) mol/l) and 4-aminopyridine (4-AP; 10(-3) mol/l). Calcium-induced activity of the isolated rat uterus pretreated with the same concentration of L-NAME, MB, or propranolol modified the kinetic of PS-induced relaxation without changes in EC(50) values. Pre-treatment with glibenclamide, TEA and 4-AP significantly reduce PS relaxing effect of calcium-induced activity and according to EC(50) values the order of magnitude was glibenclamide > TEA > 4-AP. PS is mixture of polyamines and may activate different signal-transduction pathways. Our results cleary demonstrate that in uterine smooth muscle PS act dominantly through potassium chanels and marginaly through beta-adrenergic receptos or nitric oxide-dependent pathways.