We report the cloning and characterization of a new ecotropic provirus encountered in a radio-induced thymic lymphoma of the C57BL/6 mouse. The provirus with an abnormally long LTR was inserted in the chromosomal DNA within the Pvt-1/MLVi-1/Mis-1 region which is a common integration site for MCF virus in mice and for Mo-MuLV in rats. This new ecotropic provirus was molecularly cloned and found to be infectious and competent for replication after transfection of murine cells. The recovered virus termed T3651/B was B-ecotropic, T-lymphotropic (in vivo) and highly leukemogenic for newborn C57BL/6 mice and for adult mice provided they were submitted to a subleukemogenic dose of irradiation. As compared to the AKV prototype N-ecotropic endogenous retrovirus, the T3651/B env proteins are only affected by few scattered point mutations. In contrast, the LTR has five repeats of enhancer sequences containing consensus motifs specific of the nuclear factors NF1-like, LVa, LVb and SEF1. Since a virus with such properties was encountered only once in 31 radio-induced tumors and isolated at a fourth tumor passage, a direct role of T3651/B virus in tumor genesis after irradiation is uncertain. Nevertheless, it is clear that T3651/B virus is a new leukemogenic retrovirus with a particular LTR structure which fits well with the model proposed by Rassart et al. (J. Virol. 58, 96-106, 1986) for the emergence of a thymotropic highly leukemogenic RadLV.