Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.