Precursor messenger RNA splicing requires multiple factors including U1, U2, U4, U5, and U6 small nuclear RNA's. The crosslinking reagent psoralen was used to analyze the interactions of these RNA's with an adenovirus precursor messenger RNA in HeLa nuclear extract. An endogenous U2-U4-U6 crosslinkable complex dissociated upon incubation with precursor messenger RNA. During splicing, U1, U2, U5, and U6 became crosslinked to precursor messenger RNA and U2, U5, and U6 became crosslinked to excised lariat intron. U2 also formed a doubly crosslinked complex with U6 and precursor messenger RNA. The U1, U5, and U6 crosslinks to the precursor messenger RNA mapped to intron sequences near the 5' splice site, whereas the U2 crosslink mapped to the branch site. The kinetics of crosslink formation and disappearance delineates a temporal pathway for the action of small RNA's in the spliceosome. Potential base pairing interactions between conserved sequences in the small nuclear RNA's and precursor messenger RNA at the sites of crosslinking suggest that the 5' splice site is defined in several steps prior to the first cleavage event.