Following the previous report, this investigation was made to ascertain the possibility of serial transferred cultivation for various leptospiral strains in the medium containing egg yolk lipid, and chromatographic technique was used to intend the separation of the growth supporting factor.Egg yolk ethanol extract was maintained at -15°C, and resulting muddy precipitate was separated from the supernatant. Both of the precipitate and supernatant portion were dialysed and divided into three fractions, respectively, in the same way: emulsified aqueous fraction (A or D), acetone soluble fraction (B or E) and acetone insoluble fraction (C or F). Each fraction was added to the modified Korthof's basal medium, as described previous report, at the concentration of 10 7 and 100 7 per ml., and the growth supporting activity for Leptospira canicola was tested.L. canicola inoculated to the medium containing emulsified aqueous fraction (A or D) of acetone insoluble fraction (C or F) could grow only limitedly, while in the medium containing acetone soluble fraction (B or E) the organisms could grow sufficiently and they could transferred serially to the third culture.The activity of the acetone soluble fraction was ascertained with other strains: L. canicola, L. autztmnalis, L. hebdonuzdis, L. australis, L. pomona and L. icterohaenzorrhagiae. Only two among the eight strains, L. canicola and L. australis, could grow sufficiently and transferred serially up to the tenth culture successfully.Consequently, an attempt to separate the growth supporting substances found in the acetone soluble fraction was carried out by use of silica gel column chromatography. Seven or eight chromatographic fractions were yielded and their activities for L. canicola were tested. The growth supporting activity of each fraction, however, decreased or lost, and even when all the fractions were mixed according to their yields, the restoration to the original activity was not obtained.