The number of tubules/mm2 (tubule density) of horse hoof horn was quantified in samples taken from the left forefeet of 8 randomly selected slaughterhouse horses in order to establish the normal tubule density characteristics at the midline dead centre (MDC) for the stratum medium of horse hoof. In the past the measurement of tubule distribution within the hoof has lacked objectivity. The horse hoof tubule density results are compared to a recent objective study carried out on pony hoof. A similar 4 zone pattern of tubule density was observed, although the precise zonal boundaries and tubule density values differed to those found for pony hoof. There were significant differences in tubule density between zones. Comparison with pony hoof revealed significant tubule density differences in zones 1, 2 and 4; however, there was no significant difference in zone 3. The existence of a 4 zoned pattern of tubule density for horse hoof, as for pony hoof, has been confirmed.