Heparin exerts an anti-proliferative effect in smooth muscle cells, and the Ca²⁺/calmodulindependent protein kinase (CaMK) signalling pathway is heparin-sensitive. Here we report that transfection with a truncated 326 amino acid fragment of CaMK-II increases basal activity of CaMK-II in mesangial cells. Ionomycin increased CaMK-II activity in both transfected and untransfected cells, with a concomitant increase in activated Ca²⁺/calmodulin. Heparin (1 µg/ml), but not chondroitin or dermatan sulfate, significantly attenuated both serum- or ionomycin-induced CaMK-II activity, and attendant c-fos mRNA expression, but did not affect upstream Ca²⁺/calmodulin. Autophosphorylation of Thr₂₈₆ generates an autonomously active CaMK-II. Both serum and ionomycin increased phosphorylation at this site and increased CaMK-II activity in antiphosphothreonine immunoprecipitates. Heparin (1 µg/ml) did not inhibit phosphorylation of Thr₂₈₆ (though much higher concentrations did). Replacement of Thr₂₈₆ with Asp produces a constitutively active mutant that was insensitive to ionomycin but was inhibited by heparin maximally at 1 µg/ml. These results suggest that heparin at physiological concentrations acts at or downstream of CaMK-II to suppress its activity independent of an effect on autophosphorylation.