Involvement of intracellular Ca²⁺ and extracelular signal-regulated kinases (ERK1/2) phosphorylation in the fast nongenomic effects of androgens in myotubes was investigated. Testosterone or nandrolone produced fast (less than 1 min) and transient increases in intracellular Ca²⁺, with an oscillatory pattern. Calcium signals were slightly reduced in Ca²⁺-free medium but lack of oscillations was evident. Signals were blocked by U-73122 and xestospongin B, inhibitors of inositol 1,4,5-trisphosphate (IP₃) pathway. Furthermore, IP₃ increased transiently 2- to 3-fold, 45 s after hormone addition. Cyproterone neither affected the fast Ca²⁺ signal, nor the increase in IP₃. Calcium increases could also be induced by the impermeant testosterone conjugated to BSA (T-BSA) and the effect of testosterone was abolished in cells incubated with GDPS or pertussis toxin (PTX). Stimulation of myotubes either with testosterone, nandrolone or T-BSA increased immunodetectable phosphorylation of ERK1/2 within 5 min and this effect was not inhibited by cyproterone. Phosphorylation was blocked by use of BAPTA-AM, U-73122 and xestospongin B as well as by dominant negative Ras, MEK or the MEK inhibitor PD-98059. In addition, GDPS or PTX blocked ERK1/2 phosphorylation. These results are consistent with a fast effect of testosterone, involving a G protein linked receptor at the plasma membrane, IP₃-mediated Ca²⁺ signal and Ras/MEK/ERK pathway in muscle cells.