The effects of the natural polyamines, putrescine, spermidine and spermine on single calcium-activated potassium channels from clonal rat pituitary tumor cells (GH3) were studied. Applied to inside-out patches, polyamines were found to reduce the current amplitude and open probability of the channels in a dose- and voltage-dependent manner, indicating that polyamines act as fast blockers which sense a fraction of the electrical field in the channel pore. The Kd for spermine was 11.2 mM for the reduction of unitary current amplitude and 0.7 mM for the reduction of the open probability. The order of effectiveness was spermine > spermidine > putrescine. From fitting beta-functions to current amplitude histograms, blocking and unblocking rates were determined as 11.4 x 10(4) sec-1 and 21.9 x 10(4) sec-1, respectively. The reduction of the channel open probability was relieved by an increase of the Ca2+ concentration of the internal solution, indicating that polyamines compete with Ca2+ at the Ca2+ sensor of the channel. Putrescine antagonized the effect of spermine on the channel current amplitude. The results suggest that polyamines at intracellular millimolar concentrations suppress ion channel activity and therefore may effect electrical discharge behavior of excitable cells.