Testicular regression in rodents occurs after short-day exposure. Vascular support is withdrawn during regression, and, presumably, new angiogenesis is inhibited. Blood vessel growth and maintenance are regulated by paracrine factors, including vascular endothelial growth factor (VEGF). Reduced angiogenesis may contribute to the onset of photoperiod-induced regression; i.e., reduction of VEGF protein would be detected early during gonadal atrophy. Alternatively, loss of blood vessel maintenance may reflect reduced testicular volume. If true, VEGF would not be expected to decline until significant regression occurred. To discriminate between these hypotheses, white-footed mice (Peromyscus leucopus) were maintained in either long (LD 16:8) or short (LD 8:16) photoperiod. Immunohistochemical and Western analyses revealed high VEGF expression in Leydig and Sertoli cells in long-day housed males and reduced VEGF expression in short-day housed males. Reductions in VEGF preceded decreases in both seminiferous tubule diameter and spermatogenic activity by 6 wk and reduced testis mass by 8 wk, suggesting that changes in VEGF are not a consequence of gonadal regression and that VEGF may play a critical role in photoperiodic regulation of testicular function.