(T)ryptophan (Trp) biosynthesis and production of other related compounds from 1 mM each of indole (IND), L-serine (Ser), and IND plus Ser by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Ruminal microorganisms were anaerobically incubated at 39 degrees C for 12 h. Trp and other related compounds produced in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. B, P, and BP suspensions were not able to produce Trp when incubated with only IND or Ser. Appreciable amounts of Trp (9.8, 3.1, and 6.6% of substrate) were produced from IND plus Ser after 12 h by B, P, and BP suspensions, respectively. Trp produced from IND + Ser in B was found only in the hydrolyzate, whereas it was found in the medium as a free form in P. after a 12-h incubation period. Rumen bacteria and protozoa were separately demonstrated for the first time to produce Trp from IND plus Ser, and the ability of P to produce Trp from IND plus Ser was about one-third that of B in 12 h. Trp produced from IND plus Ser by B, P, and BP suspensions was simultaneously degraded into its related compounds, and, among them, indoleacetic acid (IAA) was a major product found in B. Production of IAA was 4.3, 0.3, and 3.2% of IND in 12 h by B, P, and BP suspensions, respectively. A small amount of skatole (SKT) (1.1 and 2.5% in B and BP, respectively) and p-cresol (CRL) (2.4 and 3.4% in B and BP, respectively) were also produced from IND plus Ser during 12-h incubation. P suspension produced no SKT or CRL from IND plus Ser in 12-h incubation. These results suggested, for the Gst time that both rumen bacteria and protozoa have an ability to produce Trp, from IND plus Ser, and the ability was higher in B than in P. The ratios of Trp produced From IND plus Ser to that from indolepyruvic acid by B, P, and BP were 1:3.4, 1:14.2, and 1:6.6 during 12-h incubation period. From these results, the degree of importance of producing Trp from IND plus Ser in the rumen was indicated.