Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5' end of the 5-kb RNA to a consensus splice donor site and localized the 3' end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5' splice donor site that defines the 5' end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV.