ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. LTR-env-gene-spliced mRNA of 9 and 3.5 Kb is widely expressed in human tissues and cells, but gag-pol mRNA has not been found. Further, the env gp70 gene contains an open reading frame throughout its length and its expression has recently been detected as a full-length protein. The highest expression of ERV3 detected so far is in placenta and the lowest in cytotrophoblasts and choriocarcinoma cell lines. In this report we have studied ERV3 mRNA and protein expression in the human monoblastic cell line U-937 during differentiation into monocytes/macrophages. Differentiation of U-937 cells was induced by 1,25a-dihydroxyvitamin D3 (vitD3), retinoic acid (RA), gamma interferon (IFN-gamma) and phorbol-myristate-acetate (PMA-TPA). The expression of ERV3 env mRNA was found to be differentiation-associated, with high expression detected in the late stages of monocytic development. Using TPA, the expression of ERV3 env was detected as 9- and 3.5-kb transcripts by Northern blotting, as mRNA by in situ hybridization and as a cytoplasmic 65-kDa protein by immunofluorescence and Western blots. Low levels of basal expression were found, with up-regulation of both message and protein at 24 to 48 hr after addition of TPA. Induction with vitD3, IFN-gamma and RA produced higher levels of mRNA at earlier time points. It is concluded that the U-937 cell line represents an excellent model system for further studies to study the relationship between ERV3 expression and cellular differentiation.