For determination of 3 muscle fiber types in equine skeletal muscle, a comparison of 2 preincubation buffers, each followed by myosin adenosine triphosphatase staining, was made. Serial sections of the muscle samples (n = 75) were preincubated in an acid buffer (pH 4.6) or a formaldehyde-glycine buffer (pH 7.25) and then were stained for myosin adenosine triphosphatase. Differentiation of muscle fibers into type I, IIA, and IIB was identical with both techniques; however, in the samples prepared at pH 4.6, type I fibers were black; type IIA, light gray; and type IIB, dark gray. In the samples prepared at pH 7.25, types I, IIA, and IIB fibers were white, light gray, and dark gray respectively. The formaldehyde-glycine preincubation buffer (at pH 7.25) gave more consistent results, was easier to prepare, and retained cytoarchitecture better, compared with the samples prepared at pH 4.6.