In this study, single and double-labeling immunofluorescence histochemistry, Western blot and real-time polymerase chain reaction were used to study the expression of P2Y6 receptors in developing mouse skeletal muscle and during injury and repair. The results show that P2Y6 receptor immunoreactive (ir) cells were first detected in the dermamyotome at embryonic (E) day 9. The number and immunostaining intensity of the P2Y6 receptor-ir cells increased from E9 to E13, but decreased from E15 to postnatal day 60 in the developing skeletal muscle system. The expression levels of P2Y6 receptor protein and mRNA increased rapidly from 1 to 5 days after skeletal muscle injury and then decreased almost to the control level from 7 to 10 days, at the beginning of regeneration. P2Y6 receptor-immunoreactivity was mainly localized to the ends of single myoblasts and myotube processes in the developing and injury-repair skeletal muscle tissues. These data suggest that the P2Y6 receptor may be involved in the development and regeneration of skeletal muscle, especially in the migration and extension of the myoblast and myotube in developing and regenerating skeletal muscle.