Histochemical features of cultured rat muscle were compared to those of cultured chicken muscle, as well as of young embryonic and regenerating rat muscle. Primary cultures were established from trypsin-dissociated myoblasts of thigh muscle from 17- to 19-day-old rat embryos and of breast muscle from 10- to 12-day-old chicken embryos. Histochemical reactions for myofibrillar adenosine triphosphatase (ATPase) performed at pH 9.4 and for ATPase after acid pH 4.35 preincubation, phosphorylase, succinic dehydrogenase, reduced diphosphopyridine nucleotide dehydrogenase and myoglobin were applied to examine cultured muscle, gastrocnemius muscle of 15-17 day-old embryos and regenerating muscle fibers in cold-injured gastrocnemius muscle of adult rats. Cultured rat and chicken muscles could be best distinguished by the ATPase reaction following acid preincubation (positive in cultured rat muscle, negative in cultured chicken muscle) and by a different pattern of phosphorylase staining. However, the histochemical features characteristic for cultured aneural rat muscle were also present in young regenerating and embryonic rat muscles before innervation and differentiation of fiber types have occurred in these muscles. It seems, that young, aneural myotubes, regardless of the environment in which they develop, maintain their histochemical features, probably characteristic for the species.