In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit. L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay. In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture. Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one. Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner. Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA. However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule. L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4x10(6)/M. L17 bound the pre-rRNA molecule preferentially (Ka=17x10(6)/M) but also bound the mature 25S rRNA species (Ka=10x10(6)/M). L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well (Ka=11-12x10(6)/M). L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with Ka=3.1 vs. 2.5x10(6)M, respectively. We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.