In an earlier study of the distribution of O-serotypes among clinical isolates of Serratia marcescens, two apparently new serotypes were identified, represented by strains S1254 and S3255. Studies using ELISA, immunoblotting and the Quellung reaction have shown that they qualify for inclusion in the O-antigenic typing scheme on three counts: (1) they possess chemically distinct O-antigenic repeating units, (2) the O-antigens are serologically distinguishable from all others, and (3) they are found in a significant proportion of clinical S. marcescens strains (13% and 6% respectively), S1254, the type strain for serotype O27, is an acapsular strain which expressed a glucorhamman with a disaccharide repeating unit as its lipopolysaccharide side chain. It cross-reacts with serotype O4, the O antigen of which is an O-acetylated form of the O27 glucorhamnan, but this cross-reaction can be eliminated by reciprocal cross-absorption. S3255, the type strain for serotype O28, has a mannose homopolymer as its O-antigen and is the only S. marcescens serotype with a trimeric repeating-unit structure. However, it cross-reacts with the O5 serotype strain due to similarities in their acidic capsular polysaccharides. Cross-absorption and the production of serum to an acapsular variant of serotype strain O28 produced typing reagents which could differentiate serotypes O5 and O28.