AbstractWe examined histoenzymatic characteristics of human muscle fibers grown in monolayer culture and innervated de novo in culture for 60–90 days by fetal rat spinal cord neurons. Serial cryostat cross sections were obtained using a freshly frozen sandwich of adult rat muscle and cultured human muscle. An Advanced degree of morphologic and histoenzymatic maturation of cultured human muscle was reached after innervation. In contrast to aneurally cultured human muscle fibers, the innervated muscle fibers were smaller in diameter and had myonuclei preferentially located at the periphery of the fiber. The innervated fibers contained a well‐developed intermyofibrillar network revealed by the NADH‐TR and SDH reactions. Phosphorylase activity was strong to moderate in most muscle fibers. Although most of the innervated cultured muscle fibers were still not fully differentiated into two histochemical fiber types because they had strong ATPase activity after both alkaline and acid preincubation, a few of them had an ATPase profile similar to type 2 fibers in human adult muscle and had reciprocal staining with phosphorylase and NADH‐TR reactions. This is the first evidence of differentiation into different histochemical fiber types of human muscle cultured in monolayer and innervated de novo by fetal rat spin