The effect of the gene Po on the activity of chalcone isomerase was investigated in Petunia hybrida. Furthermore, isomerase activities isolated from petals were compared with those extracted from anthers. No effect of Po on the pH-dependence of the isomerase and its kinetic properties was observed. With respect to these criteria, the enzyme extracted from anthers behaved in an identical manner to that extracted from petals. Upon chromatofocussing of a petal extract two peaks of activity were present with slightly different isoelectric points (pI 4.8 and pI 5.1). The occurrence of these activities was dependent on the method of enzyme extraction. An isolation procedure using polyvinylpyrrolidone besides Dowex to remove phenolic compounds, followed by (NH4)2SO4 precipitation of the protein, resulted in only one peak of isomerase at a pI of 5.3. This observation was independent of Po and did not occur in anthers. In anthers one peak of enzyme activity with a pI of 4.5 was present. The moleuclar weight of the isomerase from flowers (62,500 dalton in Po-dominant and Po-recessive plants) differed from the molecular weight of the anther enzyme (44,000 dalton). In Po-recessive mutants the isomerase activity in mature flowers was low compared with Po-dominant mutants, indicating that the mutation in Po either reflects a temporal mutation in the expression of chalcone isomerase or an increased degradation of the enzyme.