Summary This study was designed to characterize further 88 streptococci of serological group L, isolated from bovines, pigs, poultry and humans. Most group L streptococci from bovines grew as granular sediment with long chains in fluid media and with compact colonies in soft agar. Most group L streptococci from pigs grew with a uniform turbidity in fluid media, short chains, and with diffuse colonies in soft agar. The biochemical characteristics, determined with the Strepzym identification system, revealed no significant differences between the group L streptococci of various origins. A positive opacity factor reaction could mainly be observed with group L streptococci from bovines and humans, but was less pronounced with group L streptococci from pigs and poultry. In addition, 27 group L streptococci reacted with type antigen X or R and 9 cultures with M1 or M6 specific antiserum. The determination of antibiotic resistance patterns revealed that all group L streptococci were resistant to tetracycline and minocycline, part of the cultures were resistant to chloramphenicol, erythromycin and gentamicin, and all cultures were susceptible to penicillin, bacitracin, cefoxitin and nitrofurantoin. All these data allowed an individual characterization of group L streptococci, possibly useful for epidemiological studies.