Anti-alkaline phosphatase antibody (AP Ab) was specific in 9% of 249 anti-acetylcholine receptor (AChR) Ab-positive myasthenia gravis (MG) (SPMG) patients but not in patients with AChR Ab-negative MG (SNMG), other neurological and immunological diseases, or healthy volunteers. No cross-reactivity and no significant titer correlation were found between AP Ab and AChR Ab. We confirmed immunologically by radioimmunoassay and western blot analysis the presence of antibodies directed against AP. AP Ab-positive SPMG patients were characterized clinically as having female predominance and a more severe form of generalized MG than AP Ab-negative SPMG patients, and about half required artificial ventilation at maximum severity. AP Ab's pathogenic role in MG is yet unclarified, but our findings show AP to be a novel antigen among the various autoantigens present in MG patients and in whom AP Ab may modify clinical symptoms.