The three-dimensional HPLC system was used to detect the presence and determine the levels of biogenic monoamines, including precursor amino acids and metabolites, simultaneously in an extract of the ciliated protozoan, Tetrahymena pyriformis. 2. Representative biogenic monoamines, such as dopamine (DA), 5-hydroxytryptamine (5-HT), epinephrine (E) and kynurenine (KYN) were found to be synthesized in Tetrahymena and released into the growth medium. 3. The following metabolic pathways were suggested to be operative: (L-DOPA)-DA-(Epinine)-E-dihydroxyphenylethyleneglycol (DOPEG)-vanillylmandelic acid (VMA) and tyrosine-4 (TYR-4)-tyramine (TYRA)-hydroxyphenylacetic acid-4 (HPAC-4) in the case of catecholamines, and tryptophan (TRP)-5-HT and TRP-KYN-xanthurenic acid (XA) in the case of indolalkylamines. 4. As judged from the released metabolites, systems for generation of biogenic monoamines in Tetrahymena seem to be active during the logarithmic phase of its growth.