The possible participation of calmodulin in the activation of store-operated Ca2+ entry (SOC) in single rat hepatocytes was investigated microspectrofluorimetrically. SOC was triggered after discharging intracellular Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin in the absence of external Ca2+. Re-admission of bath Ca2+ caused a rapid and pronounced Ca2+ entry. The calmodulin antagonists calmidazolium or CGS 9343B applied before the thapsigargin treatment inhibited SOC, whereas they were ineffective when added after the thapsigargin-induced Ca2+ transient. This study suggests that activation of calmodulin after the elevation of cytosolic Ca2+ associated with the emptying of Ca2+ stores is involved in the triggering of SOC in hepatocytes.