The effect of platelet activating factor (PAF) on the intracellular cytosolic levels of free calcium ([Ca2+]i) was studied in cultured epithelium from cow trachea. In fura-2-loaded cells, PAF (10(-9) to 10(-5) M), but not lyso-PAF, increased [Ca2+]i in a concentration-dependent manner, from 106 +/- 15 to 270 +/- 40 nM (P < 0.05). This [Ca2+]i response consisted of a transient increase that peaked within 15 s after addition and a subsequent sustained elevation that reached a plateau after 1 min. The potency for the sustained response was greater by approximately 1 log U than that for the transient response. Preincubation of the cells with the PAF receptor antagonist CV6209 (10(-6) M) inhibited the increase in [Ca2+]i. Ca(2+)-free medium (2 mM EGTA) totally abolished the sustained response to PAF, but it only partially inhibited the transient response. Verapamil (10(-5) M) also largely inhibited the sustained response. Moreover, PAF transiently increased inositol triphosphate (IP3) levels, peaking 10 s after addition. These data suggest that (1) a PAF-induced increase in [Ca2+]i is mediated via PAF receptors, (2) PAF causes both transient [Ca2+]i release from intracellular stores through IP3 production and sustained [Ca2+]i influx from extracellular solution, and (3) Ca2+ influx may be a major pathway of PAF-induced increase in [Ca2+]i in cow tracheal epithelium.