The aim of the present study is to test whether mechanical strain uniquely regulates muscle fibre atrophy/hypertrophy and adaptation of the number of sarcomeres in series within mature muscle fibres in vitro. Mature single muscle fibres from Xenopus laevis illiofibularis muscle were cultured (4 - 97 days) while kept at negative strain (∼20% below passive slack length, 'short fibres') or at positive strain (∼5% over passive slack length, 'long fibres'). Before and after culture the number of sarcomeres in series was determined using laser diffraction. During culture, twitch and tetanic force characteristics were measured every day. Survival time of long fibres was substantially less than that of short fibres. Of the long fibres 40% died or became inexcitable within 1 week, whereas this did not occur for short fibres. During culture, twitch and tetanic force of all short fibres increased substantially. Regression analysis showed that the post-culture number of sarcomeres in series was not significantly changed compared to the number before culture. It is concluded that culture at negative strain does not result in atrophy or a reduction of the number of sarcomeres in series, even after 97 days. For the long fibres we did not detect any hypertrophy as tetanic force remained stable or decreased slowly, while twitch force varied. Regression analysis of the change of the number of sarcomeres in series as a function of the culture time showed a positive slope (P = 0.054). Two out of four long fibres that were cultured for at least 2 weeks showed an increase in the number of sarcomeres of 4 - 5%. Compared with in vivo adaptation to mechanical stimuli this is much less than would be expected. The data suggest that strain may not be the only factor that regulates hypertrophy and the number of sarcomeres in series. © 2004 Kluwer Academic Publishers.