The unusual nucleoside, N-[ 9-(p-D-ribofuranosyl)purin-6-ylcarbamoyl] threonine (VA), has been found in transfer-RNA (tRNA) of many species (42). It has been located at the 3'-end of the anticodon of several specific tRNA's (3-5). In the course of our investigations on the function and biosynthesis of VA ( 6) we have found it necessary to develop a microassay that would allow quantitation of this nucleoside in 1 mg or less of tRNA. Previously, about 100 mg tRNA was necessary for quantitative isolation and optical analysis of VA (1). A tritium-labeling technique has been described by Randerath and Randerath (7-9) for subngnomolar analysis of ribose derivatives. The present communication describes a modification of this procedure to provide a facile method for pmole quantitation of VA in tRNA. MATERIALS [3H] -Sodium borohydride (sp.act. 2175 pCi/pmole) , Aquas&t II, adenosine, cytidine, guanosine, inosine, and uridine were obtained from Chemical and Radioisotope Division of International Chemical and Nuclear Corporation. N1-Methylguanosine, N2-methylguanosine, N2,iVdimethylguanosine, N1-methyladenosine, N7-methylguanosine, and &methylcytidine were purchased from Cycle Chemical Co. Dihydrouridine was from P-L Biochemicals. /3-Pseudouridine was purified (10) from an anomeric a$ mixture obtained from Sigma Chemical Company. We are grateful to the following members of the Institute for gifts of nucleosides: N6-methyladenosine, 2'-0-methyladenosine, 2'-O-methylguanosine, and 4-thiouridine from Dr. R. K. Robins; 5-carboxymethyluridine (cm5U) and 5-carboxymethyluridine methyl ester from G. Ivanovics; and VA (synthesized by the urethane method of Chheda and Hong (11)) from