We determined whether protein kinase C (PKC) isoforms are redistributed and phosphorylated in response to acute exercise in skeletal muscle. Muscle biopsies were obtained from six healthy subjects (four women, two men; age 25 +/- 1 years) before, during, and after 60 min of one-leg cycle ergometry at approximately 70% VO(2peak). Exercise for 30 and 60 min was associated with a three- and fourfold increase in PKC-zeta/lambda abundance and a four- and threefold increase in phosphorylation, respectively, in total membranes (P < 0.05) and a decrease in PKC-zeta/lambda phosphorylation in cytosolic fractions. During exercise recovery, PKC-zeta/lambda abundance and phosphorylation remained elevated. PKC-zeta/lambda abundance and phosphorylation were increased in nonexercised muscle upon cessation of exercise, indicating a systemic response may contribute to changes in PKC abundance and phosphorylation. Exercise did not change PKC-delta or -epsilon abundance or phosphorylation in either the cytosolic or total membrane fraction. In conclusion, exercise is associated with an isoform-specific effect on PKC. PKC-zeta/lambda are candidate PKC isoforms that may play a role in the regulation of exercise-related changes in metabolic and gene-regulatory responses.