The phosphate transfer system of Haseltine et al. , consisting of a ribosomal wash obtained from a stringent strain of Escherichia coli , washed ribosomes, GTP, and ATP, was used to prepare large quantities of guanosine tetra- and pentaphosphates, the magic spot compounds MS I and MS II of Cashel and Gallant. In our hands, the Haseltine et al. system yielded predominantly guanosine tetraphosphate, ppGpp. This system was used exclusively in the described experiments, with ATP labeled with 32 P in the β- and γ-positions as donor. The β-label was found to produce a ppG p and the γ-label a ppGp . 0001000 1101011 0011100 0011100 1101011 0001000 0000000 0101100 1110010 0100001 0100001 0100001 0110010 0101100 0100000 0100000 1110000 .--> Furthermore, [ 3 H]GDP + [γ- 32 P]ATP yielded ppGpp in a 3 H: 32 P ratio of 1:1. The results indicate a transfer of the terminal pyrophosphoryl group of ATP as a unit. The position of the transferred pyrophosphoryl was assayed for by preparation of pG from ppG p with Zn ++ -activated inorganic pyrophosphatase from yeast. The pG was then assayed with 3′-nucleotidase, which liberated practically all the labeled phosphate. This result indicate that the phosphate transfer from ATP to GDP yields guanosine 5′-diphosphate-3′-diphosphate.