Hybridization experiments were conducted to detect nucleic acid homology among recognized species of lactobacilli. Tritiated complementary ribonucleic acid (RNA) was synthesized and reacted with homologous and heterologous deoxyribonucleic acid (DNA) preparations. The amount of radioactivity retained by nitrocellulose membrane filters indicated the degree of DNA–RNA interaction. The results were discussed in relation to a previous study in which lactobacilli were placed in three groups on the basis of the guanine plus cytosine (GC) content of the DNA. Group I; a 42% or greater reaction was observed when Lactobacillus helveticus tritiated RNA was hybridized with DNA from L. helveticus, L. bulgaricus, L. jugurti, and L. (jugurti) bulgaricus. The latter two strains appeared to be closely related and were easily distinguished from L. helveticus. No appreciable cross reaction occurred with L. helveticus tritiated RNA and DNA from L. salivarius. Group II; no DNA–RNA hybrid formation was evident when L. casei tritiated RNA was reacted with DNA from L. plantarum, L. brevis, or L. buchneri; however, reactions of 50 to 100% were observed for nine strains of L. casei. Group III; L. lactis appeared to be more closely related to L. leichmannii than to L. delbrueckii. Also, L. fermenti and L. cellobiosus constituted a distinct subgroup within group III. The results presented substantiate and further define the grouping based on GC content previously proposed. Further subdivision of each group was suggested. The possibility of a phylogenetic relationship between selected lactobacilli and Bacillus coagulans was discussed also.