A procedure was developed for the biosynthetic preparation of "N-labelled guanosine and inosine through the action of a mutant Bacillus subtilis strain. Crude [N', 1,3,7,guanosine and [1,3,7,9-''N]inosine were isolated from the culture filtrate by precipitation and anion-exchange chromatography (Scheme 1 ). No cell lysis and no enzymatic degradation was necessary. The per-isobutyrylated derivatives 1 and 2 were isolated from a complex mixture, purified by virtue of their different lipophilicity, and separated in three steps involving normaland reversed-phase silica-gel chromatography. One litre of complex nutrient medium yielded 8.44 mmol of guanosine derivative and 2.84 mmol of inosine derivative with high average ''N enrichment (83.5 and 91.9 atom-%, resp.). [N6,1,3,7,9-"N]Adenosine (4) was obtained from 2',3',5'-tri-O-is0butyryl[l,3,7,9-'~N]inosine (1) through the ammonolysis of its 1,2,4-triazolyl derivative with aqueous "NH, (Scheme 2).Introduction. -The advent of multidimensional heteronuclear 'H-NMR spectroscopy for the elucidation of the solution structure of large biomolecules [l] [2] led to a number of investigations aiming at the preparation of defined "N-labelled RNA fragments [3] [4]. The general approach was a biosynthetic one: Microorganisms grown on minimal media containing ['5NJammonium salts were used to produce labelled RNA. After enzymatic degradation to the nucleosides and enzymic phosporylation, all four "N-labelled ribonucleoside 5'-triphosphates were obtained in 1-100 pmol amounts per litre of culture medium. Those were used as substrates for an enzymic in vitro RNA synthesis by T7 RNA polymerase.Our approach divides the preparation of "N-labelled ribonucleosides into a biosynthetic and a synthetic one, with the aim to obtain g-quantities of the nucleosides for their use in both in vitro and chemical [U-I5N]RNA synthesis. Here, we describe the preparation of the purine nucleosides [ l,3,7,9-'5N]inosine and [N2, 1 ,3,7,9-'SN]guanosine. They were directly obtained from a conventionally cultivated mutant Bacillus subtilis strain NA6601 [5]. Under certain growth conditions, these Gram -positive bacteria are capable of releasing large quantities of guanosine (G) and inosine (I) into the growth medium. This greatly simplifies the isolation and purification procedure. Subsequently, [ 1,3,7,9-''PJlinosine was chemically converted into [N6, 1,3,7,9-"N]adenosine. Results andDiscussion. -Production, Isolation, and Purification of the Main Metabolites. Optimal aerobic growth of B. subtilis NA6601 required a medium based on sorbitol and autolyzed yeast (SD medium) for precultures and a complex medium based on I) Diploma thesis ( A . N . 1991, M . M . and T.E. 1992, 0. B. 1993).