Microbial assimilation of 3 amino acids (glutamic acid, alanine, and ornithine) was characterized in 3 lakes and 2 marine stations using the Michaelis-Menten kinetic approach. The calculated Kt + Sn concentrations were related to chemical concentration measurements of dissolved free amino acids (DFAA) to evaluate the biological and the chemical determinations of the DFAA pools. Concentrations of Kt + Sn always were larger than chemical measurements of the Sn concentrations. Kt + Sn and Sn varied from 11.5 and 9.5 nM (alanine, oligotrophic lake) to 288.7 and 89.9 nM (ornithine, marine harbor station), respectively. Subtracting Sn from the Kt + Sn concentrations, Kt was found to range from 12-897% of the chemically measured Sn concentrations. To test whether the DFAA actually were free, dissolved molecules, dissolved material in the water samples was separated into various molecular size classes by means of gel permeation chromatography. From 47-116% of the DFAA in the untreated water samples was recovered in the low molecular fraction (<700 Daltons). Variation in recoveries mainly appeared to be due to an incomplete chromatographic separation and difficulties in obtaining proper blank levels. The present observations suggest that labeled tracers can be used in the study of DFAA assimilation and that the DFAA are free, dissolved molecules. This partly conflicts with previously published reports.