Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7.deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.The modified nucleoside 7-{5-[(4,5-dihydroxy-2-cyclopent-1-yl)amino]methyl}-7-deaza-guanosine, queuosine (Q), and its glycosylated derivatives (Q*) are found in the first position of the anticodons for tRNAs accommodating the amino acids asparagine, aspartic acid, histidine, and tyrosine (reviewed in reference 25). Q is widely distributed in the biosphere, occurring in the tRNA of plants, animals, and eubacteria (25). Bacteria synthesize Q de novo by the irreversible, base-for-base exchange of 7-aminomethyl-7-deazaguanine for guanine in position 34 of the tRNA, after which the cyclopentenediol moiety is added in an unknown fashion (25). The base exchange is catalyzed by tRNAguanine ribosyltransferase (25). Eucaryotes synthesize Q similarly, with the exception that queuine, the base of Q, is exchanged for the guanine in position 34 (22,23,25,31). Animals do not synthesize Q de novo and must obtain queuine from the diet or gut flora, either as the free base or, after degradation of Q-containing (Q+) tRNA, by the specific cleavage of Q-5'-phosphate (Q-5'-P) to queuine (12,18,21,28). It is notable that queuine is not a substrate for Escherichia coli tRNA-guanine ribosyltransferase (25).Animal serum provides queuine to mammalian cells in tissue culture, and this has been exploited to develop a sensitive bioassay for queuine: in the mouse cell line L-M, which is propagated in a serum-free medium, the Q content of tRNA is directly related to the concentration of queuine added to the medium (21, 22). The L-M cell assay has enabled the detection of significant amounts of free queuine in plant and animal products common to the human diet (21). * Corresponding author. Whether plants synthesize queuine de novo or obtain it from exogenous sources has not been established.The present study was prompted by the discovery that extracts from Chlorella pyrenoidosa, which had been cultivated in a defined queuine-f...