This study examined effects of an enzyme preparation (Depol 740L, Biocatalyst, Pontypridd, UK) containing high esterase (32 U/ml) activity on in vitro digestibility and ruminal in situ degradability of three tropical grass hays. The enzyme was applied at 0, 0.5, 1, 2 and 3 g/100 g dry matter (DM) to ground (1 mm), 12-week regrowths of Pensacola bahiagrass (BAH), Coastal bermudagrass (C-B), and Tifton 85 bermudagrass (T-85) hays in a 3 × 5 factorial experiment replicated thrice. Twenty-four hours later, samples were incubated in triplicate for 6, 24 and 48 h in buffered ruminal fluid in two ANKOM Daisy II incubators The enzyme was also applied at similar rates to ground (4 mm) samples of hays and incubated in duplicate in each of two cows for 0, 3, 6, 9, 12, 24, 48, 72, 96 and 120 h. Enzyme application enhanced (P<0.05) release of WSC from the hays. The 6 and 24 h in vitro dry matter digestibility (IVDMD) of the hays increased linearly (P<0.05) as the enzyme application rate increased. Incremental enzyme addition increased (P<0.05) release of free ester and ether-linked ferulic acid from BAH and T-85, respectively, as well as ester-linked p-coumaric acid from T-85 and BAH. The 48-h IVDMD of C-B increased linearly (P=0.05) as enzyme application increased (487, 484, 489, 506, 510 g/kg) but that of T-85 and BAH did not. The 6-h neutral detergent fiber (NDF) digestibility (IVNDFD) of T-85 increased linearly (P<0.05) with increasing enzyme addition (41, 34, 29, 61, 72 g/kg), whereas that of C-B or BAH did not. In vitro 24-h IVNDFD of hays increased linearly (P<0.01) as enzyme application increased. Incremental enzyme addition increased (P<0.05) the in situ washout fraction of all hays, and the potentially degradable fraction, the extent of degradability, and the effective degradability of C-B. Enzyme application increased (P<0.05) the degradation rate of T-85 and decreased (P<0.01) the lag phase of C-B. This enzyme enhanced in vitro digestion and ruminal in situ degradation of hays, but the pattern and extent of improvement was forage specific.