Regulation of gene expression of prolactin gene still a point of study for many Scientifics so this work was designed to investigate the transcriptional activity of 3- deletion of the rat prolactin gene promoter. A plasmid containing the rPrl gene promoter (-423/+38) was restricted at the unique EcoR1 site of 3' deletion of the ePrl promoter sequences in rPrl (-423/+38). With suitable condition established prPrl (-423/+38) was EcoR1 restricted in preparative amounts and Bal31 treated for optimized time periods and the resulting DNA fragments pooled. The selected positives were then restricted with BamH1, Hind III and the presence of a Bam H1/ Hind III fragment 423 bp verified by agarose gel electrophoresis. The results shows that the most proximal high affinity Pit-1/GHF-1 binding element is not an absolute requirement for GC cell specific transcriptional activity and that the Pit-1/GHF-1 binding elements with sequence to -423, is sufficient to confer relatively high transcriptional drive, in orientation independent manner