Abstract (1) The effect of the potent non-steroidal antiandrogen, flutamide, on neural receptor binding of testosterone and intermale aggressive behavior was studied. (2) The main findings were: (i) flutamide exhibited a high affinity in vivo for androgen binding macromolecules within the hypothalamus and preoptic-septal region of the brain. This finding was in contrast with the negligible testosterone receptor antagonist activity of flutamide in vitro; (ii) flutamide, given in conjunction with testosterone to castrated male mice, had no suppressive effect on the restoration of intermale aggressive behavior. (3) The relative effectiveness of flutamide in suppressing [ 3H]-testosterone binding in vivo and in vitro is interpreted to mean that flutamide exerts its antiandrogenic effect within the central nervous system, at least in part, by means of its conversion in vivo to an active metabolite. (4) The results of the study on the effects of flutamide on aggressive behavior are interpreted in support of the aromatization hypothesis for the mechanism of action of testosterone on the central nervous system.