We examined the role of testosterone (T) in regulating the weight of the songbird syrinx and the activity of two cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (AChE). Castration of adult male zebra finches or neonatal canaries results in a lowering of syringeal weight and CAT and AChE activity. Administration of T for 1-4 weeks restores syringeal weight and AChE to intact levels in male zebra finches. Activity of CAT in muscle is not fully restored. Ovariectomy of female zebra finches and canaries does not affect these syringeal parameters, but T administration to ovariectomized females for 1 month increases syringeal weight and AChE activity. In the zebra finch tracheosyringealis nerve, activity of CAT and AChE is decreased one month after castration. T administration to castrates maintains nerve AChE activity but not CAT. In contrast to changes in the syrinx, tongue muscles do not change in weight or enzyme activity when circulating T levels are altered. Effects of muscle use and disuse were found on syringeal weight and AChE activity, but an androgenic effect also operates in addition. Results suggest that one mechanism for T regulation of singing in passerine birds is through induction of specific enzymatic proteins in androgen target neurons and muscles.