This study reports on the optimization of the culture conditions for a thermotolerant eukaryotic algal strain, Prototheca zopfii RND16, which can effectively degrade and assimilate n-alkanes at elevated temperatures. RND16 was able to grow on 1% (v/v) n-alkanes (C-14-C-17) at temperatures up to 38degreesC. This ability differs from a previous finding that P. zopfii did not grow on hydrocarbons under temperature conditions above 25degreesC. Increasing the temperature from 25degreesC to 30-35degreesC resulted in an increase in the rate of n-alkane consumption during growth of RND16 in quiescent culture. In shaking culture at 35degreesC, RND16 removed a 1% n-alkane mixture almost completely at the basal medium salinity within 8 d. However, an appreciable decrease in the extent of hydrocarbon utilization was observed with increasing salinity and substrate concentration in the medium. The slow consumption of the n-alkanes in the early stage of degradation at 25degreesC was improved by supplementing 0.1% glucose. A comparative study on the nuclear small subunit rDNA (18S rDNA) sequences of three strains within the genus Prototheca revealed that both P. zopfiii ATCC 30253, which utilize n-alkanes at room temperature, and P. moriformis ATCC 50081, which does not assimilate n-alkanes, are closely related to RND16.