The maximal shortening velocity of a muscle (V_max) provides a link between its macroscopic properties and the underlying biochemical reactions and is altered in some diseases. Two methods that are widely used for determining V_maxare afterloaded and isotonic release contractions. To determine whether these two methods give equivalent results, we calculated V_maxin 9 intact single fibres from the lumbrical muscles of the frog Xenopus laevis (9.5–15.5 °C, stimulation frequency 35–70 Hz). The data were modelled using a 3-state cross-bridge model in which the states were inactive, detached, and attached. Afterloaded contractions gave lower predictions of V_maxthan did isotonic release contractions in all 9 fibres (3.20 ± 0.84 versus 4.11 ± 1.08 lengths per second, respectively; means ± SD, p = 0.001) and underestimated unloaded shortening velocity measured with the slack test by an average of 29% (p = 0.001, n = 6). Excellent model predictions could be obtained by assuming that activation is inhibited by shortening. We conclude that under the experimental conditions used in this study, afterloaded and isotonic release contractions do not give equivalent results. When a change in the V_maxmeasured with afterloaded contractions is observed in diseased muscle, it is important to consider that this may reflect differences in either activation kinetics or cross-bridge cycling rates.