The role of Ca2+-dependent phospholipase A(2) (PLA(2)) in the mechanism of skeletal muscle damage in broiler chickens was examined in vitro using a novel, synthetic, PLA(2)-specific inhibitor Ro31-499/001 (Ro31). Muscle damage was assessed by measurement of creatine kinase (CK) efflux from isolated muscles into the incubation medium. Treatment with the specific Ca2+-ionophore 4-Br-A23187 (5 muM) caused a 72% elevation (P < 0.05) in muscle Ca-45(2+) accumulation, which was associated with a marked increase (P < 0.001) in muscle CK efflux (7.6-fold). Incubation with Ro31 (50 muM) reduced (P < 0.001) CK efflux from muscles treated with ionophore (45%) but was without effect on Ca-45 accumulation. Treatment with the Na+ ionophore monensin (100 muM) induced 55% (P < 0.05) elevation in Ca-45(2+) accumulation with a concomitant 2.5-fold increase (P < 0.001) in CK loss. Muscles incubated with monensin in the presence of Ro31 exhibited a 49% reduction (P < 0.001) in CK leakage but showed no change in Ca-45(2+) uptake. The results indicate that increasing external Ca2+ entry, directly or indirectly, and elevation of intracellular Ca2+, significantly alters sarcolemmal integrity resulting in increased CK efflux from broiler skeletal muscle. This process is, at least in part, dependent upon activation of PLA(2) activity and thus inhibitable by Ro31. It is further proposed that muscle damage in poultry induced by a range of stresses, and insults may also be mediated by a Ro31 sensitive, PLA(2)-dependent component. The findings have implications for strategies to reduce or prevent myopathies in poultry affecting bird welfare and product quality.