The primary sequence of the long unique region L-DNA (L for low GC) of rhesus monkey rhadinovirus (RRV) isolate 26-95 was determined. The L-DNA consists of 130,733 bp that contain 84 open reading frames (ORFs). The overall organization of the RRV26-95 genome was found to be very similar to that of human Kaposi sarcoma-associated herpesvirus (KSHV). BLAST search analysis revealed that in almost all cases RRV26-95 coding sequences have a greater degree of similarity to corresponding KSHV sequences than to other herpesviruses. All of the ORFs present in KSHV have at least one homologue in RRV26-95 except K3 and K5 (bovine herpesvirus-4 immediate-early protein homologues), K7 (nut-1), and K12 (Kaposin). RRV26-95 contains one MIP-1 and eight interferon regulatory factor (vIRF) homologues compared to three MIP-1 and four vIRF homologues in KSHV. All homologues are correspondingly located in KSHV and RRV with the exception of dihydrofolate reductase (DHFR). DHFR is correspondingly located near the left end of the genome in RRV26-95 and herpesvirus saimiri (HVS), but in KSHV the DHFR gene is displaced 16,069 nucleotides in a rightward direction in the genome. DHFR is also unusual in that the RRV26-95 DHFR more closely resembles HVS DHFR (74% similarity) than KSHV DHFR (55% similarity). Of the 84 ORFs in RRV26-95, 83 contain sequences similar to the recently determined sequences of the independent RRV isolate 17577. RRV26-95 and RRV17577 sequences differ in that ORF 67.5 sequences contained in RRV26-95 were not found in RRV17577. In addition, ORF 4 is significantly shorter in RRV26-95 than was reported for RRV17577 (395 versus 645 amino acids). Only four of the corresponding ORFs between RRV26-95 and RRV17577 exhibited less than 95% sequence identity: glycoproteins H and L, uracil DNA glucosidase, and a tegument protein (ORF 67). Both RRV26-95 and RRV17577 have unique ORFs between positions 21444 to 21752 and 110910 to 114899 in a rightward direction and from positions 116524 to 111082 in a leftward direction that are not found in KSHV. Our analysis indicates that RRV26-95 and RRV17577 are clearly independent isolates of the same virus species and that both are closely related in structural organization and overall sequence to KSHV. The availability of detailed sequence information, the ability to grow RRV lytically in cell culture, and the ability to infect monkeys experimentally with RRV will facilitate the construction of mutant strains of virus for evaluating the contribution of individual genes to biological properties.