The Y chromosome of Drosophila melanogaster accounts for approximately 13% of a normal male genome and is entirely heterochromatic. It carries six genes required exclusively for spermatogenesis. Here we report a novel activity of the Y chromosome that regulates gene expression in primary spermatocytes. By examining the expression of a reporter gene in X/Y and X/O males, we show that a specific region of the Y long arm carries a trans-activator that regulates transcription in spermatogenesis. In the absence of the Y trans-activator, the level of the reporter expression is greatly reduced in primary spermatocytes and the expression pattern is restricted to young primary spermatocytes. Further analysis shows that the Y trans-activator is dispersed in the h1-h10 region on the Y long arm and is functionally redundant, indicating involvement of the repetitive sequences on the Y chromosome. In addition, the Y trans-activator appears to act in a tissue-specific manner, functioning only in the male germ line. We propose that the Y trans-activator plays an important role in regulating gene expression during spermatogenesis.