Forty-three independent variants of the Novikoff hepatoma cell line have been isolated for their ability to use D-xylose, D-ribose, and/or L-arabinose as a sole carbon and energy source. The variants exhibited marked morphological changes and a loss or decrease of cloning efficiency in soft agar. The xylose and arabinose variants showed similar phenotypes while the ribose variants were a phenotypically heterogenous group. Two major classes of variants were found with regard to their specificity for pentoses: one class could grow on ribose, xylose, or arabinose, while the second class grew only on ribose. The lack of specificity for pentose use was correlated with the ability to use pentitols for growth. The frequency of pentose-utilizing clones was 5 X 10(-2) to 10(-3), and nitrosoguanidine treatment increased this frequency tenfold. Fluctuation analyses showed the appearance of pentose-utilizing variants to be a random event. Of the variants examined, 84% expressed a stable pentose phenotype, and of those, 6% were cold sensitive and 8% were temperature sensitive for pentose utilization. In addition to the suggested mutational basis for the pentose phenotype, two variants showed a large increase in chromosome number from 73 +/- 3 to 132 +/- 10.