We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in pre-meiotic S-phase by neutral/neutral 2D gel-electrophoresis. The comparison of origin activity of each origin in mitotic and pre-meiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in pre-meiotic S-phase. ARS605 is located within the ORF of Msh4 gene that is transcribed specifically during an early stage of meiosis. Systematic analyses of relationships between Msh4 transcription and ARS605 origin activity revealed that transcription of Msh4 inhibited the ARS605 origin activity by removing ORC from ARS605. Deletion of Ume6, a transcription factor responsible for repressing Msh4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.