286 We have previously shown that the activation of the 70-KDa S6 kinase(p70S6K) is correlated with in vivo skeletal muscle hypertrophy. In order to determine whether the stimulus for p70S6K activation has a mechanical component, we performed a study on cultured murine skeletal muscle cells, in which the electrical and chemical inputs could be controlled. C2C12 myoblasts were seeded in silastic membrane wells, and cultured for 1 week until branching, multinucleated myotubes had formed. These myotubes were subjected to a computer generated stretching regimen consisting of sixty uniaxial stretches. The stretches were grouped into ten sets of six 12% stretches over 20 seconds with a 50 second rest interval. The cells were harvested immediately, 3 hours, or 24 hours following stretch. Immediately following stretch, there was a small increase in p70S6K activity (13.7±16%, NS). As with the in vivo model, maximal activation of p70S6K was not seen until 3 hours following stretch (75.9±16.6% increase) and was maintained elevated at 24 hours (58.4% increase). The time course of p70S6K activation suggested that an autocrine mechanism may be involved in the activation of p70S6K. To test this hypothesis, unstretched C2C12 myotubes were incubated with medium from the 3 hour post-stretch group for 10 minutes. Application of the conditioned medium resulted in the activation of p70S6K in unstretched myotubes (82.4% increase) to levels comparable to the 3 hour stretch group. These data indicate that p70S6K is activated in C2C12 myotubes in response to a mechanical stimulus, via a mechanism that most likely involves an autocrine signaling pathway.