We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5′ splice site (5′SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5′SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5′SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5′SS RNA when the APA is placed within the 5′ exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5′SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5′SS junction (positions −2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5′ end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5′SS interaction. Unexpectedly, thio-modifications within the region of the 5′SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.