The molecular mechanism underlying photoperiodism is not well understood in any organism. Long day-induced conversion of prohormone thyroxine (T₄) to bioactive triiodothyronine (T₃) within the mediobasal hypothalamus (MBH) is critical for the photoperiodic regulation of reproduction. However, since thyroidectomy does not completely block the photoperiodic response in some species, the existence of a thyroid hormone-independent regulatory mechanism appears certain. To identify this novel mechanism, differential subtractive hybridization analysis was performed using MBH of quail kept under short day and long day conditions. This analysis identified a gene encoding transforming growth factor (TGF). Expression of TGF mRNA was induced in the median eminence by the stimulus of long days and this induction was observed at dusk on the first long day. This rapid induction of TGF mRNA was similar to induction of the thyroid hormone-activating enzyme gene (Dio2), which is the earliest event yet determined in the photoinduction process. Expression analysis of epidermal growth factor (EGF) receptors revealed strong expression of erbB4 and weak expression of erbB1 and erbB2 in the median eminence. Intracerebroventricular infusion of physiological dose of TGF induced luteinizing hormone (LH) secretion and testicular growth under short day conditions. Finally we demonstrate that T₃ implantation and TGF infusion into the MBH, either of which causes testicular growth, do not affect the expression of TGF and Dio2, respectively. Thus, long day-induced activation of the TGF signalling pathway appears to mediate a thyroid hormone-independent pathway for the photoperiodic regulation of reproduction.