The lipoteichoic acid from Lactococcus lactis Kiel 48337 was analyzed. It had 61% of its glycerophosphate residues substituted with alpha-D-galactopyranosyl residues. Non-substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage. The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE-Sephadex into 10 pairs of molecular species with n from 1 to 10. The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents. A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid. Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern. The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P = 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.