Altered queuine modification of tRNA has been correlated to neoplastic transformation, but no direct cause and effect relationship has been defined. In the present study, a potential role for this alteration has been assigned. The tRNA in normal Chinese hamster embryo cells is significantly more queuine modified than the tRNA in their transformed Chinese hamster embryo counterparts, even though the specific activity of the queuine modification enzyme is much lower in Chinese hamster embryo cells than in transformed Chinese hamster embryo cells. Substrate availability appears to be responsible for the queuine hypomodification of tRNA in the transformed cells, since addition of excess exogenous queuine to the culture medium results in incorporation of queuine into the anti-codon of the undermodified tRNAs. Most importantly, the excess queuine inhibits anchorage-independent growth of transformed Chinese hamster embryo cells, thereby implicating queuine hypomodification of tRNA in the expression of this transformed phenotype.