T cells recognizing the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) are increased in multiple sclerosis (MS), and there are elevated numbers of T cells recognizing the nicotinic acetylcholine receptor (AChR) in myasthenia gravis (MG). However, the cytokine repertoires in these diseases are largely unknown. We adopted in situ hybridization with radiolabeled complementary DNA oligonucleotide probes to enumerate mononuclear cells that expressed the T-helper type 1 (Th1) cell-related interferon-gamma (IFN-gamma) and Th2-associated interleukin-4 (IL-4) after short-term culture in the presence of autoantigen. High numbers of IFN-gamma and IL-4 mRNA-expressing cells in response to MBP and PLP were detected in patients with untreated MS, and to AChR in MG. The levels of IFN-gamma and IL-4 mRNA-positive cells in MS after culture in the presence of AChR, and in MG after culture in the presence of MBP or PLP, did not differ from those detected after culture without antigen. The CSF of MS patients contained four- to eightfold more myelin protein-reactive IFN-gamma and IL-4 expressing cells. The findings imply that MS and MG are associated with mixed Th1- and Th2-like cell responses directed to organ-specific target antigens.