We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139-151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89-101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108-120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89-101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108-120 develop encephalomyelitis similar to the disease induced by the cognate peptide. We demonstrate that NAD 108-120 induces T cells that cross-react with MBP 89-101; the antigen-sensitized T cells, which produce predominantly T helper (T(h)) 1 and T(h)17 cytokines, transfer disease in naive SJL recipients reminiscent of the disease induced with MBP 89-101. This is the first report to demonstrate that a solitary microbe can induce CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.