The frequency of gap junction formation between neurons and myoblasts of chick embryo leg skeletal muscle changes as a function of the developmental stage of the muscle. Cells from leg muscle of various ages and states of innervation were first cloned in vitro and then co-cultured with ciliary ganglion, spinal cord, or dorsal root ganglion neurons. The presence of gap junctions between cells was identified by the passage of fluorescent dyes or electric currents from one cell to another. The clones examined were fusing muscle clones (myoblasts) as well as nonfusing clones. Mononuclear cells of either kind of clone derived from legs younger than stage 24 (E4) dye-couple with other clone cells but do not dye-couple with neurons. Myoblasts of fusing muscle clones derived from stage 24 through stage 29 (E5) legs dye-couple with neurons at high frequency; nonfusing clones from these same embryos do not contain cells that dye-couple with neurons. Mononuclear cells of both fusing and nonfusing clones from normally innervated stage 30 (E6) through 38 (E12) legs do not dye-couple with neurons at significant frequencies. Additionally, aneural legs of denervated E10-E12 embryos yield muscle clones in which the myoblasts again dye-couple with neurons at high frequency. The ability to form communicating junctions between muscle cells and neurons in culture is restricted to myoblasts cloned from legs of those stages of development and conditions of innervation that define and limit neuron-dependent alteration of myoblast populations in vivo.