A microcytotoxicity assay to prove directly against myelin basic protein directed cytotoxic T cells has been developed. Spleen and lymph node cells of myelin basic protein immunized strain 13 guinea pigs were restimulated in vitro with basic protein for 5 days and used as attacking cells. Autologous spleen cells were coated with about 10(7) molecules basic protein by hydrophobic attachment, labelled with 51Cr and used as targets. Concentration dependent lysis (74% corrected lysis) was measured with attackers gained 12 days post immunization. Controls performed using unspecific (PHA) stimulated attackers and non BE coated target showed no lysis. Lysis was shown to be MHC restricted. The test may serve as a tool for the assay of human demyelinating diseases.