We investigated the role of viral transcripts of human T-lymphotropic virus type I (HTLV-I) in the cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMCs) of patients with human T-lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). To detect the HTLV-I p40tax protein, we developed a new sensitive method of immunohistochemistry combined with tyramide signal amplification and quantitative analysis. Seven patients with HAM/TSP were examined. As controls, four patients with other neurological diseases were examined; two of these patients were infected with HTLV-I and the other two were not. Both the CSF cells and PBMCs were reacted with a monoclonal antibody, Lt-4, for p40tax protein, followed by secondary antibody labeled with horseradish peroxidase. This was visualized by fluorescein directly labeled with tyramide and the number of positive cells was quantified with a Laser Scanning Cytometer. In the samples from patients with HAM/TSP, the HTLV-I p40tax protein was successfully detected by tyramide signal amplification, but not without it. In HAM/TSP patients, 0.04-1.16% of the CSF cells and 0.02-0.54% of PBMCs were positive for the HTLV-I p40tax protein, respectively. The expression of the HTLV-I p40tax protein in the CSF cells was more frequent than that in PBMCs in both HAM/TSP patients and HTLV-I carriers, and was also more frequent in the patients with HAM/TSP of shorter duration of illness. This technique could be a powerful tool to investigate the pathogenic mechanism of diseases associated with HTLV-I.