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Melatonin is an important rhythmic endocrine signal within the circadian system of mammals. The hypophyseal pars tuberalis (PT) is a major target for melatonin and shows a high density of melatonin type 1 receptors (MT1). Melatonin affects expression of clock genes in PT cells which encode for transcriptional regulators of rhythmic gene expression. In this study, microarray analysis was performed to screen for genes coding for transcriptional regulators under the control of MT1 receptors in the mouse PT. Gene expression levels were compared between melatonin-proficient mice deficient for MT1 (MT1−/−) and the corresponding wild-type (WT) during mid-subjective day (CT06, low endogenous melatonin levels) and mid-subjective night (CT18, high endogenous melatonin levels). In situ hybridization was used to validate the data obtained by microarray analysis to analyze the acute effect of daytime melatonin application on gene expression in the wild-type PT. In the wild-type PT, expression of Tim was higher during day as compared to night. These day/night differences in gene expression were not observed in the PT of MT1−/− mice, demonstrating the impact of MT1-mediated signal transduction pathway. Day-time application of melatonin acutely affected Tim and Cry1 expression but not Neurod1 and Npas4 expression. We conclude that melatonin regulates expression of genes coding for transcriptional regulators in the PT through MT1 receptors by either acute or long-term mechanisms.