Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.