The polycations spermine, neomycin and polylysine potentiated Ca(2+)-activated force in beta-escin permeabilized guinea-pig ileum strips. The effect was inhibited by the calmodulin antagonists trifluoperazine, mastoparan and W13. Potentiation was slow or absent in chi-toxin permeabilized strips, indicating dependence on penetration of the polycations into cells. The effects of spermine and neomycin were maintained after extensive permeabilization by beta-escin, which eliminated the contractile effect of GTPgammaS. Replacement of ATP by CTP, which is not a substrate for myosin light chain kinase, inhibited contractile potentiation. Potentiation of Ca(2+)-activated contractions was associated with increased phosphorylation of the myosin regulatory light chains (LC20). A contractile effect of polylysine and neomycin was also seen in Ca(2+)-free medium and after partial LC20 thiophosphorylation, indicating that phosphorylation-independent processes may contribute to the response. Although spermine does not cause contraction in Ca(2+)-free medium at physiological [MgATP], it did so when [MgATP] was lowered to 40 micron. Similar to high-[Mg2+], the rate of contraction on addition of ATP to strips incubated with microcystin-LR in inhibit phosphatase activity was increased by the polycations, but only at [Ca2+] < 0.3 micron. The results suggest that polycations increase Ca(2+)-activated force by inhibiting myosin phosphatase activity, thereby increasing myosin LC20 phosphorylation. However, additional activation mechanisms, evident at low [Ca2+] and at low [ATP] and possibly involving direct activation of myosin, contribute to their effect.