Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered human gamma herpesvirus (HHV-8) that plays an important role in Kaposi's sarcoma development. Here, we further characterize the regulation of the early HHV-8 gene, open reading frame 57 (ORF57). ORF57 is a spliced gene consisting of two exons with a 108-bp intron near the 5' end. The ORF57 mRNA can potentially be initiated at two different start sites, and its expression can be significantly stimulated by ORF50, an HHV-8 immediate early gene. The target site for ORF50 transactivation was mapped to a 40-bp fragment compassing nt 81904 to 81943 in the ORF57 promoter. Our study on the regulation of ORF57 expression by ORF50 provides the basis for further studies on the regulation of HHV-8 lytic gene expression.