19F-n.m.r. spectroscopy was used to study the binding of 3',5'-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to 'flip' about its symmetry axis, and the rate (7.3 X 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.