Adult skeletal muscle fibers can be isolated and cultured but tend to dedifferentiate and sprout with time in culture. We examined isolated adult mouse flexor digitorum brevis muscle fibers under various culture conditions by monitoring maintenance of the same fibers at 2-d intervals using survival analysis. Fibers plated on laminin and cultured in serum-free media did not show sprouting and exhibited significantly (P < 0.0001) longer survival (median survival time, T₅₀ = 10.2 d) than fibers in serum-containing media (T₅₀ = 3.3 d). Cell proliferation was markedly suppressed in serum-free cultures. Multiple or delayed Ca² transients in response to brief field stimulation were often observed in dedifferentiated fibers after several d in serum-containing media but were not observed in fibers in serum-free media. The addition of cytosine arabinoside to serum-containing cultures did not prolong fiber survival (P = 0.39) and did not eliminate sprouting but did greatly suppress proliferation of nonmuscle cells. Fibers cultured in agarose gel with serum exhibited small, bud-like extensions but no sprouts and did not survive as long (T₅₀ = 6.2 d) as fibers plated on laminin and cultured in serum-free media (T₅₀ = 10.2 d) did. These results demonstrate that both morphological and physiological properties of fibers become modified in serum-containing media but can be retained by culturing without serum.