Using chemical and enzymatic structure-specific probes adapted to rapid gel sequencing techniques, we have analyzed the solution conformations of precursors to two yeast tRNAs which contain an intervening sequence, pre-tRNAPhe and pre-tRNATyr. Interpretation of the data was greatly facilitated by performing direct mature/precursor tRNA comparisons. In addition, the effects of tertiary interactions on probe specificity could be evaluated from the results obtained with mature tRNAPhe, whose crystal structure is known. We find: 1) the folding of the precursor CCA terminus, acceptor stem, T psi C stem, variable loop, anticodon stem, and D stem identical with that of the equivalent regions in the cognate, mature tRNA. 2) The T psi C loop and D loop appear to vary slightly in tertiary structure between mature and precursor species. 3) The precursors contain a helix involving the anticodon triplet and a complementary sequence in the intron. 4) The stability of this helix is much greater for pre-tRNAPhe than for pre-tRNATyr. 5) The splice sites for both precursors are located in single-stranded loops. These results bear out predictions based on genetic analyses and are consistent with the view that recognition of universally conserved features of tRNA structure allows all tRNA precursors containing intervening sequences to be processed by a single splicing apparatus.