Human T‐cell lymphotropic virus type I (HTLV‐I)‐associated myelopathy (HAM) and tropical spastic paraparesis belong to a new group of neurological diseases associated with retroviral infection. An HTLV‐I‐like virus has recently been implicated in multiple sclerosis as well. We studied paired cerebrospinal fluid and serum specimens from HAM and multiple sclerosis patients by isoelectric focusing and an isoelectric focusing HTLV‐I p24 overlay technique to clarify the role of HTLV‐I in these diseases. We detected oligoclonal bands by isoelectric focusing with silver‐staining in cerebrospinal fluid, but not serum, from all 5 HAM and all 9 multiple sclerosis patients. An isoelectric focusing HTLV‐I p24 overlay technique demonstrated anti‐p24 antibody in HAM cerebrospinal fluid at a different pI distribution than that seen in paired serum, indicating local synthesis of anti‐p24 antibody within the central nervous system. Oligoclonal bands in HAM cerebrospinal fluid corresponded in pI distribution to anti‐p24 antibody activity, suggesting the presence of an ongoing HTLV‐I infection in the central nervous system. Multiple sclerosis patients had no evidence of anti‐HTLV‐I activity by p24 radioimmunoprecipitation assay, Western immunoblots, or isoelectric focusing HTLV‐I p24 overlay analysis. Our data support a role for HTLV‐I as an etiological agent in HAM, but not in multiple sclerosis.