The use of chemical ionization mass spectrometry for the characterization of analogues of methotrexate, N-[4-[[(2,4diamino-6-pteridinyl)methyl]methylamino~~nzoyl~~-glutamic acid, has been studied. With methane as reactant gas, abundant (MH]+ ions were generally not produced. However, with ammonia, especially in conjunction with thermal desorption from a platinum wire, significant amounts of [MH]+ ions were formed by methotrexate, the a-, y-and diamide analogues, and series of a-and y-monoalkylamide derivatives. The 1-butyl esters of the various monoamides behaved similarly to the corresponding monoamides, except for the ready loss of isobutylene. Fragment ions from both methane and ammonia chemical ionization were formed by cleavages 'benzylic' to the pteridine ring, by bond breakage a t the amide bond between the aminobenzoyl and glutamyl moieties, and by fragmentations on both sides of this amide bond. Fragment ions from these processes, in conjunction with further disintegration of the glutamyl moiety, are diagnostic of the structures of the pteridine, aminobenzoyl and glutamyl moieties of the analogues. Examples of application to structural determination are given.