Virus-like 30S (VL30) elements are endogenous retro-elements of the mouse retrotransposon family. These elements are transcriptionally responsive in a temporal and tissue-specific manner due to the U3 promoter region of the elements' long terminal repeat (LTR). We have analyzed VL30 promoters from erythroid progenitor cell lines (MEL 585S and ELM-I-1) that contrasted in their response to erythropoietin (epo). Through RT-PCR-generated cDNAs, VL30 promoters were identified and showed homology to the third and fourth U3 subgroups, with GATA-1, Jak2/STAT5, and B10 RRE sites. One clone (ELM5) showed 97% homology to BVL-1, a putative epo-responsive VL30 element. In addition, a novel U3 promoter (MEL/ELM CONSTIT) showed complete sequence homology between both cell lines. Ribonuclease protection confirmed that epo-induced VL30 promoters were activated in ELM-I-1 cells, whereas the conserved VL30 MEL-ELM CONSTIT VL30 promoter showed no enhanced expression in the epo-unresponsive MEL cells. Identification of these U3 promoters suggests that VL30s are conserved and can be transcriptionally activated in an epo-specific manner.