The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2, 4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 microg of DCP-C ml(-1). A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 microg of C ml of DCP(-1) and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 microg of C ml(-1) was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 microg of C ml(-1) was not degraded in mixed culture even by a high density, 10(5) CFU ml(-1), of DP-4. When glucose was added to this mixed culture to a final concentration of 1 microg of C ml(-1), the initial concentration of 0.01 microg of C ml of DCP(-1) was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 microg of C ml(-1). The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 microg of C ml of DCP(-1) was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.