Effect of pertussis toxin treatment on the actions of insulin-like growth factor-I (IGF-I) was examined in Balb/c 3T3 cells. In competent cells primed with epidermal growth factor, IGF-I stimulated both calcium influx and DNA synthesis. When these primed competent cells were treated with various concentrations of pertussis toxin for 2 hrs, IGF-I-induced calcium influx and DNA synthesis were inhibited in a similar dose dependent manner. The inhibitory action of pertussis toxin well coincided with the toxin-induced ADP-ribosylation of a 41 K-Da protein. These results suggest a possible involvement of a pertussis toxin-sensitive GTP-binding protein in the action of IGF-I.