Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene residue attached to the 3'-termini, in the presence of cognate Phe-tRNA(Phe) has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.