4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.