The structure of phycoviolobilin, the photoactive chromophore of a-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a △3, 3 1-double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-K-phycoerythrocyanin (PecA). In the absence of the components of K-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (λmax= 641nm) and low photoactivity (＜10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (λmax=565nm) and high photoreversible photochromism (100% type I) has been identified as integral K-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.