A new technique, which uses formation of double derivatives for the detection of hydroxy acids in spent cultural media by gas–liquid chromatography (g.l.c), is described. The method is practical enough for use in routine analysis. Fourteen species of the genus Neisseria, including several serogroups of N. meningitidis, were tested. Four hydroxy acids and a keto acid which have not been previously reported in Neisseria were tentatively identified from acidified ether extracts of spent cultural media on both polar and nonpolar columns. Different g.l.c. profiles were obtained for several serogroups of N. meningitidis, N. gonorrhoeae, N. haemolysans, N. lactamicus, N. perflava, N. flava, N. mucosa, N. catarrhalis, N. ovis, and N. caviae. Strains of N. meningitidis, N. subflava, N. flavescens, N. caviae, and N. catarrhalis comprise a heterogeneous group. One strain previously identified as N. caviae gave a g.l.c. profile like that of N. ovis. The data indicate that g.l.c. analysis of metabolic products provides information necessary for differentiating many members of the genus.