Galectin-3 (M(r) approximately 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted of galectin-3 by affinity adsorption on a lactose-agarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control cellobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.