1. Mechanical factor(s) associated with the initiation of eccentric contraction-induced muscle injury were investigated in isolated rat soleus muscles (n = 180; 42 protocols with 4-6 muscles per protocol). Five eccentric contractions were performed with 4 min between contractions. Three levels of peak eccentric contraction force (100, 125 and 150% of pre-injury maximal isometric tetanic tension, P0), length change (0.1, 0.2 and 0.3 muscle length, L0) and lengthening velocity (0.5, 1.0 and 1.5 L0/s) were utilized. Force was varied with stimulation frequency (10-150 Hz). The eccentric contractions were initiated at muscle lengths of 0.85 or 0.90 L0. Following the fifth eccentric contraction, the muscle was incubated in Krebs-Ringer buffer for 60 min. Peak isometric twitch tension (PT), P0, maximal rate of tension development (+ dP/dt), maximal rate of relaxation (-dP/dt), and creatine kinase (CK) release were measured prior to the five eccentric contractions and at 15 min intervals during the incubation period. Total muscle [Ca2+] was measured after 60 min incubation. 2. The mean (+/- S.E.M.) initial decline in P0 for the muscles performing the most injurious protocol was 13.6 +/- 4.8% (n = 6); P0 in control muscles immediately following performance of five isometric contractions was elevated 1.2 +/- 1.0% (n = 8). These means were different at probability, p = 0.005. Mean [ATP] in muscles immediately following the isometric control and most injurious protocols, respectively, were 16.30 +/- 1.49 and 19.84 +/- 1.38 mumol/g dry wt (p = 0.229). 3. Decrements in P0, PT, +dP/dt, and -dP/dt immediately after the injury protocol were related most closely to the peak forces produced during the eccentric contractions; greater initial declines in P0, +dP/dt and -dP/dt were also observed at higher lengthening velocities independent of peak force. Slow declines in P0 and -dP/dt during the 60 min incubation following the injury protocol were greatest for muscles performing contractions at the longer initial length. CK release was independent of all mechanical factors with the exception of lengthening velocity. CK activity at 45 and 60 min into the incubation period was greater for muscles lengthened at the highest velocity used (1.5 L0/s). Mean total muscle [Ca2+] for muscles performing the eccentric contractions was elevated by 38% over isometric control muscles but the elevation was unrelated to any of the four mechanical factors. 4. These data support the hypothesis that eccentric contraction-induced injury is initiated by mechanical factors, with muscle tension playing the dominant role.(ABSTRACT TRUNCATED AT 400 WORDS)