Stiff-man syndrome (SMS) is a rare disorder of the central nervous system thought to result from an impairment of gamma-aminobutyric acid (GABA)ergic neurotransmission. Autoantibodies to the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), present in about 60% of SMS patients, have suggested an autoimmune pathogenesis of SMS. By using serum or cerebrospinal fluid from 25 SMS patients, we assessed the effect of GAD autoantibodies (GAD-A) on GAD enzymatic activity in vitro; 83% of GAD-A-positive SMS sera reduced GABA production in crude rat cerebellar extracts, whereas GAD-A- sera from SMS patients or healthy blood donors did not alter the enzyme activity. Inhibition of GABA synthesis by SMS sera was dose dependent and mediated by the purified IgG fraction of the sera. Human monoclonal GAD65-A and IgG purified from serum of GAD-A-positive patients with insulin-dependent diabetes or autoimmune polyendocrine syndrome did not affect GAD activity, suggesting that a specific epitope recognition of GAD-A mediates inhibition of GAD. The disease-specific detection of GAD-inhibitory antibodies is compatible with their functional involvement in the etiopathology of SMS; the relevance of such antibodies in vivo, however, remains to be determined.