Effects of the aminoglycoside spectinomycin on the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) have been investigated. The kinetic analysis demonstrated that spectinomycin acts as a mixed noncompetitive inhibitor for the td intron RNA with a K-i of 7.2 mM. Increasing the spectinomycin concentration raised the K-m values with the corresponding decrease of V-max and k(cat) values. The specificity of the splicing inhibition by spectinomycin is due to changes in both K-m and k(cat). The splicing inhibition by spectinomycin is dependent on pH changes and Mg2+ concentration, indicating electrostatic interactions with the intron RNA. It has been proposed that the key structural features in spectinomycin responsible for the inhibition of splicing may be the hydroxyl groups on the antibiotic.