The purpose of this study was to determine the normal variation in the number of uranaffin-positive organelles in normal human platelets and to study the effect of pH and fixation on the uranaffin reaction. The normal variation in uranaffin organelles was determined by studying platelets from nine normal subjects. At pH 3.9, the mean number of reactive sites/platelet profile was 0.43 +/- 0.10 when platelets were fixed with lower glutaraldehyde concentrations and 0.56 +/- 0.24 with higher glutaraldehyde concentrations. Fixed platelets were reacted at pH 2.8, 3.9, 5.0, and 7.0 with four different uranaffin procedures that varied in the extent of fixation and rinse steps (isotonic saline vs. cacodylate buffer). The number of uranaffin-positive sites in 200 platelet profiles was scored under the electron microscope. There was a progressive increase in the number of reactive sites/platelet profile as the pH increased from 2.8 to 7.0. In general, higher pH favored granule matrix and core staining, whereas low pH favored both the staining of granule membranes and their contents. The uranaffin reaction showed organelle specificity when run under certain experimental conditions. At low pH and using isotonic saline in the rinse steps, only the dense bodies and ribosomes stained. The biochemical content of the dense body responsible for uranaffin reactivity is discussed.