Adenosine at low concentration is toxic to mammalian cells in culture. This may escape notice because some sera (such as calf or human) commonly used in culture media, contain adenosine deaminase. In the absence of serum deaminase, adenosine produced inhibition of growth of a number of established cell lines at concentrations as low as 5 × 10 − 6 M, and killed at 2 × 10 −5 M. This effect required the presence of cellular adenosine kinase, since a mutant line deficient in this enzyme was 70-fold less sensitive to adenosine. The toxic substance is therefore derived from adenosine by phosphorylation, and is probably one of the adenosine nucleotides. The toxic effect of adenosine in concentrations up to 2 × 10 −4 M was completely prevented by the addition of uridine or of pyrimidines potentially convertible to uridine, suggesting that the adenosine was interfering with endogenous synthesis of uridylate. In the presence of adenosine, the conversion of labelled aspartate to uridine nucleotides was reduced by 80–85 % and labelled orotate accumulated in both the cells and in the culture medium. The lethality of adenosine results from inhibition by one of its nucleotide products of the synthesis of uridylate at the stage of phosphoribosylation of orotate.