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The environment of tRNA 3′-terminus in the 80S ribosomal A and P sites was studied with a tRNAAsp analogue that bears a 4-thiouridine residue (s⁴U) attached to the 3′-terminal adenosine. The tRNAAsp analogue was obtained by in vitro T7 transcription followed by crosslinking with [³²P]ps⁴Up and removal of the 3′-terminal phosphate. It was shown that the presence of the additional nucleotide at the 3′-end does not to hinder the codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in crosslinking of the analogue exclusively to 28S rRNA. The crosslinking was completely dependent on the presence of s⁴U in the tRNA analogue. Using hydrolysis of the crosslinked 28S rRNA with RNase H in the presence of deoxyoligomers complementary to various rRNA sequences, we determined that the crosslinking occurred in fragment 4302-4540 of the 28S rRNA. This fragment is evolutionarily conservative and belongs to domain V that is involved in the formation of the peptidyl transferase site in prokaryotic ribosomes. The use of reverse transcription allowed the determination of the tRNA analogue crosslinking in the P site to nucleotides U4461 and U4502, and the analogue in the A site, to nucleotides U4469 and C4507. In addition, nucleotide C4462 was crosslinked to both P site and A site-bound tRNA analogue. An analysis of the results demonstrates that environments of the tRNA 3′-termini are closely similar in both prokaryotic and eukaryotic ribosomes.