Pituitary GH secretory profiles are sex-dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex-specificity of rat liver gene expression, and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex-specificity of 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes whose expression decreased following hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes whose expression increased following hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment, and may correspond to secondary response genes. 24 DNA-binding proteins were identified as early GH response genes, of which 15 were induced and 9 were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes such as Cyp2a2 and Cyp2c13 were down-regulated within 30 min of GH pulse treatment, as determined by hnRNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.