OBJECTIVE: To develop and validate a novel instrumented treadmill capable of determining vertical ground reaction forces of all 4 limbs simultaneously in horses. SAMPLE POPULATION: Data obtained while a horse was walking and trotting on the treadmill. PROCEDURE: 18 piezo-electric force transducers were mounted between the treadmill frame and supporting steel platform to measure the actual forces at the corresponding bearing points. Each of the 18 sensor forces is equal to the sum of the unknown hoof forces weighted with the transfer coefficients of the corresponding force application points. The 4 force traces were calculated, solving at each time point the resulting equation system, using the Gaussian least-squares method. System validation comprised the following tests: determination of the survey accuracy of the positioning system, determination of the natural frequencies of the system, linearity test of the force transfer to the individual sensors, determination of superimposed forces with the treadmill-integrated force measuring system (TiF) in a static configuration, and comparison of vertical ground reaction forces determined simultaneously by use of TiF and force shoes mounted on the forelimbs of a horse. RESULTS: Comparison between static test loads and TiF-calculated forces revealed deviations of < 1.4%. Force traces of TiF-calculated values and those recorded by use of the force shoes were highly correlated (r > or = 0.998). CONCLUSIONS AND CLINICAL RELEVANCE: This instrumented treadmill allows a reliable assessment of load distribution and interlimb coordination in a short period and, therefore, is suitable for use in experimental and clinical investigations.