A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.