The enzyme dihydrofolate reductase (DHFR) is an essential component of cellular metabolism. It is the target for a number of drugs, some of which are directed towards the bacterial form of the enzyme and function as antibiotics. Trimethoprim (TMP) is one such compound. Unfortunately, bacteria quickly evolve variants of DHFR that are both active and resistant to TMP. We have used directed evolution to mimic this evolutionary process, and have produced mutants that are both active and bind TMP with very low affinity. Analysis of these mutants reveals a great deal about DHFR as well as providing a great deal of useful information for designing new drugs.