Carbonic anhydrase (CA) activities were studied in soluble extracts and cryostat sections of skeletal muscles from prepubertal and postpubertal rats. Acetazolamide inhibition was utilized to distinguish between activities of the acetazolamide-sensitive (CA I and II) and acetazolamide-resistant (CA III) forms of the enzyme. The inhibition studies indicated that fast-twitch oxidative-glycolytic muscle fibers contained both the sensitive and resistant forms of CA. Acetazolamide-sensitive activity was localized within muscle fibers, axons, myelin, and capillaries. Axoplasmic staining was restricted to subpopulations of myelinated axons in both the dorsal and ventral roots. Soleus muscles exhibited significantly greater activity of CA III than extensor digitorum longus muscles at all ages examined. CA III was richest in slow-twitch oxidative and intrafusal fibers. During puberty, soleus muscle fibers matured and converted from fast-twitch oxidative-glycolytic to slow-twitch oxidative fibers. There was a shift from the sensitive to the resistant form of CA; CA III activity increased about sevenfold. This activity peaked earlier in the muscles of female rats than male rats. These results demonstrated a complex distribution of CA isozymes in the neuromuscular system and pointed out that isozyme content depends on both the type of muscle and the age and sex of the animal.