Forty trimethoprim analogues in which the para substituent in the benzene ring was varied were prepared for antibacterial evaluation. All were very potent inhibitors of Escherichia coli dihydrofolate reductase. The similarity of their inhibitory activities strongly suggested that the side chains beyond the first two atoms were not in contact with the enzyme. However, among 38 ether derivatives which varied widely in their bulk and lipophilicity, very few approached trimethoprim in their broad-spectrum in vitro antibacterial activity. The 4'-methyl and 4'-ethyl analogues and the allyloxy and gamma-chloropropoxy ethers had activities fairly close to that of trimethoprim. The two ethers were chosen for further evaluation in vivo. Neither compound quite matched trimethoprim in efficacy in mice, and their half-lives, as well as that of the beta-methoxyethoxy analogue, were found to be shorter in dogs.