AbstractHerpesvirus immediate early (IE) proteins are known to play key roles in establishing productive infections, regulating reactivation from latency, and creating a cellular environment favourable to viral replication. Human herpesvirus‐6 (HHV‐6) IE genes have not been studied as intensively as their homologues in the prototype betaherpesvirus human cytomegalovirus (HCMV). Whilst the HCMV IE1 gene is relatively conserved, early studies indicated that HHV‐6 IE1 exhibited a high level of sequence variation between HHV‐6A and HHV‐6B isolates, although the observation was based primarily on virus stocks that had been isolated and propagated in vitro. In this study, we investigated the level of HHV‐6 IE1 sequence variation in vivo by direct sequencing of circulating virus in clinical samples without prior in vitro culture. Sequences exactly matching those reported for reference HHV‐6 isolates were identified in clinical samples, thus the HHV‐6 laboratory strains used in the majority of in vitro studies appear to be representative of virus circulating in vivo with respect to the IE1 gene. The HHV‐6 IE1 sequence is also conserved in reference strains that had been passaged extensively in vitro. The high degree of divergence between variant A and B type IE1 sequences was confirmed, but interestingly HHV‐6B IE1 sequences were observed to further segregate into two distinct subgroups, with the laboratory strains Z29 and HST representative of these two subgroups. Within each HHV‐6B subgroup, a remarkably high level of homology was observed. Thus the HHV‐6 IE1 sequence appears highly stable, underlining its potential importance to the viral life cycle. J. Med. Virol. 71:578–584, 2003. © 2003 Wiley‐Liss, Inc.