While androgen binding sites have been localized to motoneurons of the lateral motor column (LMC) of 10-18-day chick embryo spinal cord, they have yet to be characterized biochemically. Studies were undertaken to characterize the binding of the androgen [3H]5a-dihydrotestosterone ([3H]DHT) to spinal cord cytosols. Saturable, high-affinity binding of [3H]DHT to cytosols prepared from both 6 and 10-day spinal cords was observed. The binding component was a macromolecular species as it displayed a sedimentation coefficient of 8S upon centrifugation in sucrose gradients; proteinaceous, as binding was eliminated by heating cytosols; and displayed steroid-specific binding, as other non-androgen steroid agonists did not significantly inhibit [3H]DHT binding. The number of binding sites increased 10-fold from embryonic day 6 to day 10. The availability of testosterone and presence of androgen receptors in spinal cord motoneurons as spontaneous limb motility begins suggests a possible role for androgen-receptor-dependent gene expression in the process of target-dependent differentiation of LMC motoneurons.