Bacterial chemotactic peptides are low molecular weight peptides which stimulate a wide range of neutrophil functions following binding to specific leucocyte receptors. Formyl methionyl leucyl phenylalanine (FMLP) is the major chemotactic peptide in Escherichia coli culture supernatants. This paper reports the development and validation of a radio-immunoassay (RIA) for FMLP and its application to the analysis of formyl peptide production by enteric bacteria in vitro. The assay was moderately sensitive (10 nmol/L FMLP) and highly specific showing cross reactivity with F-met-leu-tyr, F-nle-leu-phe and F-met-met-met sequences (ID50 = 200, 100 and 250 nmol/L, respectively) but no significant cross reactivity with non-formylated or other formylated di- and tri-peptides (ID50 = 10(5) nmol/L. Culture supernatants from five species of enteric bacteria were filtered, concentrated and fractionated by reverse phase high performance liquid chromatography before RIA. All five organisms produced immunoreactive F-met peptides. A major peak of immunoreactivity co-chromatographing with authentic FMLP was found in all supernatants, but additional peaks representing more hydrophobic peptides were found in Streptococcus faecalis and Bacteroides fragilis cultures. In E. coli culture supernatants, concentration of immunoreactive FMLP increased in a linear fashion during 3 h of log phase growth reaching 31.2 nmol/L(s.e.m. = 10) with final bacterial concentrations of 3 +/- 0.73 x 10(8)/mL (n = 6). These findings extend earlier work showing production of bioactive formyl oligopeptides by different species of enteric bacteria and suggest that a RIA for FMLP will be a useful tool for investigating the production and metabolic fate of such peptides in man.