The humoral immune response against human T-cell lymphotropic virus type I (HTLV-I) in the central nervous system (CNS) compartment and in the blood was investigated by enzyme immunoassay using 16 synthetic peptides corresponding to HTLV-I core and envelope sequences. We evaluated paired samples of cerebrospinal fluid and serum from HTLV-I seropositive Japanese patients, classified as follows: HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP; n = 39), patients with spinal cord disease ascribed to either HAM/TSP or to some concomitant, HTLV-I-unrelated disease (possible HAM/TSP; n = 6) or carriers without any clinical signs of HAM/TSP (n = 15). HTLV-I-peptide-specific intrathecal antibody synthesis was found in 79% of HAM/TSP patients, but only in 20% of carriers without HAM/TSP. The group of carriers without HAM/TSP showed local synthesis for some peptides (on average 0.3 peptides per patient). In most HAM/TSP patients, however, there was a diverse intrathecal immune response to several HTLV-I synthetic peptides (on average against 3.6 peptides per HAM/TSP patient), most frequently against gag p19 100-130, env gp21 458-488, and env gp46 175-199 and 288-317. The intrathecal antibody synthesis against several HTLV-I determinants may represent a pathogenic immune response in HAM/TSP and is possibly related to the infiltration of virus-infected T-cells in the spinal cord.