Single, mature muscle fibres were dissected from Xenopus leavis m. iliofibularis and attached to a force transducer in a 20 ordm;C culture chamber. Fibres were cultured in a serum-free medium at a mean sarcomere length of 2.3 mm for 8 to 22 days. The medium was supplemented with one of the following agents (final concentrations): (1) bovine insulin (~6 nmol/L or ~400 nmol/L), (2) 0.2% bovine albumin and (3) 0.2% albumin plus insulin (~120 nmol/L). During culture with insulin only, 50% of the muscle fibres showed stable force patterns for 1 to 2 weeks, whereas the other 50% of the fibres showed a >80% reduction in tetanic force during the first week. In response to caffeine, these unstable fibres produced forces which attained 90% of the initial force, indicating an impaired excitation-contraction coupling. In the presence of albumin, all cultured muscle fibres were stable. Albumin was located at the membrane of the muscle fibres using IHC. Interestingly, muscle fibres cultured in the presence of both albumin and insulin showed increases in tetanic force and fibre cross-sectional area of 19.5±2.8% and 32.5±4.9% after 17±1.7 days, respectively (P<0.007). Hypertrophy was not observed during culture with insulin or albumin only. We conclude that albumin improves the stability of the excitation-contraction coupling and facilitates the hypertrophic effects of insulin.