Transfer RNA's isolated from Bacillus subtilis, Bacillus stearothermophilus, and chicken liver have been separated by polyacrylamide gel electrophoresis at pH 5.8 in the presence of 7 M urea. The tRNA's from each source separated into unique patterns of RNA bands. The B. subtilis tRNA's separated into 5 bands, as did the B. stearothermophilus tRNA's. The chicken liver tRNA's separated into 4 or 5 bands. In each case the location of several specific tRNA's was determined by aminoacyl-ating the bulk tRNA with one [14C]amino acid according to the method described previously (l). When the relative migration rates of tRNA's from these three sources and from Escherichia coli (l) were compared, it was noted that the tRNA's specific for each amino acid generally migrated similarly, relative to each other. A possible basis for the separation on polyacrylamide gels of RNA species of similar chain length is discussed.