Tall fescue is susceptible to injury from many acetolactate synthase (ALS) inhibitors used for broadleaf weed control in turfgrass. Florasulam is an ALS inhibitor that selectively controls broadleaf weeds in tall fescue, but the mechanisms for selectivity are not well understood. The objective of this research was to evaluate the physiological basis of tall fescue tolerance to florasulam. In greenhouse experiments, florasulam rates required to injure tall fescue 20% (I20) and white clover 80% (I80) measured 320 and 65 g ai ha–1, respectively. The I20 and I80 values of another ALS inhibitor, flucarbazone, on these species measured 33 and 275 g ai ha–1, respectively. In laboratory experiments, the time required to reach 50% foliar uptake for 14C-florasulam and 14C-flucarbazone measured 23 and 62 h for white clover, respectively, and >72 h for both herbicides in tall fescue. The half-lives of florasulam and flucarbazone in tall fescue were 15 and 40 h, respectively, whereas the half-life in white clover was >72 h for both herbicides. The concentrations of florasulam and flucarbazone required to inhibit ALS enzymes 50% in excised leaves of tall fescue measured >1,000 and 32 μM, respectively. The selectivity of florasulam for white clover control in tall fescue is associated with differential levels of absorption and metabolism between species. Tall fescue has faster metabolism and less ALS enzyme inhibition from florasulam as compared to a more injurious ALS inhibitor, flucarbazone, which contributes to the differential tolerance levels between these herbicides.