A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.