P-loop NTPases (GTPase and ATPase) are widely employed in both prokaryotes and eukaryotes to regulate various cellular processes. YchF and YihA are two highly conserved NTPases in bacteria, but their cellular roles remain elusive. Previous data revealed that the ribosome or ribosomal subunits are binding partners of these two NTPases. Here, we examined the binding preferences of Escherichia coli YchF and YihA to the 30S, 50S and 70S ribosomes in the presence of different nucleotides, and assayed whether these binding preferences were associated with the stimulation of their NTPase activities. Our data show that YchF and YihA display a strong preference for the 70S and 50S, respectively. While the 70S ribosome, but not the 50S or 30S, promotes both the ATPase and GTPase activities of YchF, YihA responds to both the 50S and 70S, with the moderate GTPase stimulation (～8.8 fold) seen in the presence of the 70S ribosome.