1. Membrane currents were recorded by a patch clamp technique in guinea-pig tracheal myocytes, using the whole cell mode with Cs(+) internal solution. 2. Both neurokinin A (NKA, 1 mu M) and caffeine (10 mM) evoked Ca(2+)-activated Cl- currents (I[Cl(Ca)]) transiently. In Ca(2+)-free bathing solution, the first application of NKA or caffeine elicited I[Cl(Ca)] but the second application of these substances failed to activate it. In addition, pretreatment with ryanodine in the presence of caffeine abolished the response to both NKA and caffeine whilst heparin (200 mu g ml(-1)) only blocked the NKA-induced response. I[Cl(Ca)] was also elicited by inositol 1,4,5-trisphosphate (IP(3)). 3. Command voltage pulses positive to 0 mV from a holding potential of -60 mV activated the voltage-dependent L-type Ca2+ current (I(Ca,L)) and late outward current. Upon repolarization to the holding potential, slowly decaying inward tail currents were recorded. The outward current during the depolarizing pulses and the inward tail current were enhanced by Bay K 8644, but completely blocked by Cd2+ or nifedipine. Replacement of external Ca2+ with Ba2+, removal of Ca2+ from the bath solution, or inclusion of EGTA (5 mM) in the patch pipette, also led to abolition of these currents, indicating that they were Ca2+ dependent, and that Ca2+ influx due to I(Ca,L) activated the currents. 4. When [Cl(-)](O) or [Cl(-)](i) was changed, the reversal potential (E(rev)) of the Ca2+-activated currents shifted, thus behaving like a Cl(-)-selective ion channel as predicted by the Nernst equation. DIDS (1 mM) completely abolished the currents, also suggesting that they were I[Cl(Ca)]. 5. NKA (1 mu M) and caffeine (30 mM) transiently activated I[Cl(Ca)], and after that both agents markedly reduced I[Cl(Ca)] induced by I(Ca,L). This is probably due to sarcoplasmic reticulum (SR) Ca2+ release induced by NKA or caffeine, followed by inhibition of the Ca(2+)-induced Ca2+ release from the SR. 6. The present results indicate that I[Cl(Ca)] can be activated by SR Ca2+ release due to NKA or caffeine (through IP(3) or ryanodine receptors) as well as by Ca2+ influx due to I(Ca,L). It also suggests that activation of I[Cl(Ca)] by NKA may be mediated by the production of IP(3), which releases Ca2+ from the SR.