From a soluble cellular fraction of maize embryos we purified to apparent homogeneity a cytoplasmic histone acetyltransferase, which matches all criteria for a B-type enzyme. Using 8 chromatographic steps, we achieved a 6700-fold purification of an enzymatically active protein with a molecular weight of approximately 90 kDa. Under denaturing conditions the protein split into 2 components which migrated at 45 and 50 kDa in SDS-PAGE, suggesting that the native enzyme is a heterodimer. The purified enzyme was characterized in terms of physicochemical and kinetic properties, and substrate specificity. It was specific for histone H4, leading to acetylation of non-acetylated H4 subspecies into the di-acetylated state in vitro. Its activity was coincident with the intensity of DNA replication in meristematic cells during embryo germination. We established an electrophoretic system under non-denaturing conditions for detection of enzyme activity within the gel matrix; in combination with second dimension SDS-PAGE the procedure allowed the unambiguous identification of histone acetyltransferase, even in crude enzyme preparations.