Escherichia coli tRNA pseudouridine 55 synthase catalyzes pseudouridine formation at U55 in tRNA. A 17 base oligoribonucleotide analog of the T-arm was equivalent to intact native tRNA as a substrate for pseudouridine 55 synthase, viz., the features for substrate recognition by this enzyme are completely contained within the T-arm. The structures and activities of mutant tRNAs and T-arms were used to analyze substrate recognition by pseudouridine 55 synthase. The 17-mer T-arm was an excellent substrate for the synthase, while disruption of the stem structure of the 17-mer T-arm eliminated activity. Kinetic data on tRNA mutants lacking single T-stem base pairs indicated that only the 53:61 base pair, which maintains the 7 base loop size, was essential for activity. The identities of individual bases in the stem were unimportant provided base pairing was intact. A major function of the T-stem appears to be the maintainence of a stable stem-loop structure and proper presentation of the T-loop to pseudouridine 55 synthase. The 7 base T-loop could be expanded or contracted by 1 base and still retain activity, albeit with a 30-fold reduction in kcat. Kinetic analysis of T-loop mutants revealed the requirement for U54, U55, and A58, and a preference for C over U at position 56. Base substitutions at loop nonconserved position 59 or semiconserved positions 57 or 60 were well tolerated. Comparison of pseudouridine 55 synthase and tRNA (m5U54)-methyltransferase revealed that both enzymes required the stem-loop structure. However, pseudouridine 55 synthase was not stringent for a 7 base loop and recognized a consensus base sequence within the T-loop, while tRNA (m5U54)-methyltransferase recognized the secondary structure of the 7 member T-loop with only a specific requirement for U54, the T-loop substrate site. We conclude that recognition of tRNA by pseudouridine 55 synthase resides in the conformation of the T-arm plus four specific bases of the loop.