A serological survey for HTLV infection identified an AIDS patient with HTLV-I/HTLV-II dual seroreactivity. Two further sequential blood samples were collected (samples A and B) for PCR, restriction fragment length polymorphism (RFLP), and sequence analyses of HTLV-I and HTLV-II strains. PCR analyses confirmed dual infection in both samples. Restriction digests of the env region amplified from sample A showed the presence of an HTLV-IIa subtype; the HTLV-II provirus was found to be defective in the pol and env regions in the second sample from this patient. RFLP analysis of the HTLV-II LTR region of both samples confirmed this finding and identified an a5/bzl restriction type. Nucleotide sequence analyses revealed full homology in the HTLV-I env and LTR regions and in the HTLV-II LTR region between the two samples. These findings document the first case of an HTLV-I/HTLV-II coinfection that was fully confirmed and characterized by means of molecular analyses.