Human herpesvirus 6 (HHV-6) isolates can be classified into two variants, A and B. Comparison of genomic sequences of these variants has highlighted sequence variability in the region spanning U86 to U100. This region includes the immediate early A (IE-A) locus that was defined as positional homologue of the major IE locus of Human cytomegalovirus (HCMV) with little recognizable sequence homologies. A 3.5 kb transcript, one of the four spliced transcripts identified in the IE-A locus, is derived from the U90/89 ORF encoding the IE1 protein. We expressed six Escherichia coli fragments spanning the HHV-6A U90/89 ORF as IE1 fusion proteins. The bacterially expressed fusion protein was used to raise monospecific polyclonal antiserum for detection and identification of the IE1 protein product(s). Using this antiserum we detected 165, 190, and > 190 K proteins in HHV-6A- and HHV-6B-infected cells and the 165 K protein in cells transfected with an IE1 cDNA construct. The IE1 proteins exhibited perinuclear and cytoplasmic localization in infected cells. There was a correlation between the expression level of IE1 and the degree of permissiveness for virus growth in various cell lines. In transient expression experiments a 140 bp fragment from the upstream IE-A region was shown to possess promoter activity. The C-terminal region of IE1 delineated by amino acids (aa) 588 to 636 showed a DNA binding activity in Southwestern blot analysis.