Protein synthesis is a highly conserved process in all living cells involving several members of the translation factor (TRAFAC) class of P-loop GTPases, which play essential roles during translation. The universally conserved GTPase HflX has previously been shown to associate with the 50S ribosomal subunit, as well as to bind and hydrolyze both GTP and ATP. In an effort to elucidate the cellular function of HflX, we have determined the kinetic parameters governing the interaction between HflX and these two purine nucleotides using fluorescence-based steady-state and pre-steady-state techniques. On the basis of these, we demonstrate that the GTPase and ATPase activity of HflX is stimulated by 50S and 70S ribosomal particles. However, given cellular concentrations of the two purine nucleotides, approximately 80% of HflX will be bound to guanine nucleotides, indicating that HflX may function as a guanine nucleotide dependent enzyme in vivo. Using a highly purified reconstituted in vitro translation system, we show that the GTPase activity of HflX is also stimulated by poly(U) programmed 70S ribosomes and that the ribosome-dependent GTPase stimulation is specifically inhibited by the antibiotic chloramphenicol, which binds to the large ribosomal subunit, but not by kanamycin, an aminoglycoside targeting the small ribosomal subunit.