We used various segments of the 5'upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from-97 to the transcriptional start site and an enhancer/silencer domain from-448 to-112. The core promoter contains a GATA site at-31 to-28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from-97 to-83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from-380 to-362,-270 to-257, and-137 to-120 as well as a negatively acting subdomain at-184 to-151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from–380 to–362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocyte lineage. The subdomains from-270 to-257 and-137 to-120 function together with the subdomain from-380 to-362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from-380 to-362 or-137 to-120 reduce transcription in megakaryocytes by 10-to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.