Here we describe a human T-cell lymphotropic virus type I (HTLV-I) seronegative patient from South India with a chronic, progressive spastic paraparesis from which HTLV-I has been isolated from peripheral blood lymphocytes. HTLV-I pol and tax viral sequences were detected in DNA from fresh peripheral blood lymphocytes (PBL) by polymerase chain reaction (PCR) and liquid hybridization techniques. Southern blot analysis of the PCR products demonstrated a low copy number of HTLV-I at the level of one viral copy per 10,000 fresh PBL. A long-term CD4+ T-cell line was established from PBL of this patient using recombinant interleukin-2, OKT3, and feeder cells. DNA from these cultured lines was amplified and portions of the HTLV-I long terminal repeat (U3), pol, env, and tax regions were sequenced (a total of 1,115 bp). The sequence data showed that the HTLV-I associated with this patient was 98.8% homologous to prototype HTLV-I. Southern blot analysis also confirmed the presence of full-length HTLV-I. These results indicate that HTLV-I can be demonstrated in an HTLV-I seronegative patient from South India with a chronic progressive neurological disorder.