Acid extracts of thapsigargin-stimulated Jurkat cells revealed both intracellular and extracellular activities stimulating Ca(2+)-dependent Cl- currents on Xenopus laevis oocytes. Chromatographic fractionation of these extracts on gel filtration separated two active fractions of M(r) approximately 600 and 400. Moreover, the M(r) 600 fraction exhibited both intracellular and extracellular activities. However, the intracellular activity was absent from extracts of unstimulated Jurkat cells, suggesting its production was stimulated by thapsigargin. The further purification of this fraction by high performance thin layer chromatography resolved a single fraction which was active only on microinjection and which required calcium entry for activation of current responses. These results suggest that a single authentic calcium influx factor can be resolved by purification from confounding activities detected in crude acid extracts.