<p>Chlorophyll (Chl) <i>b</i> is essential for maximal capacity of photosynthesis in green algae and plants. Along with broadening the absorbance spectrum, Chl <i>b</i> controls the translocation of light-harvesting complex apoproteins across the chloroplast envelope inner membrane and stabilizes light-harvesting complexes in thylakoid membranes. In the absence of Chl <i>b</i>, these Chl-binding proteins are retracted into the cytosol and degraded. A protein designated chlorophyllide (Chlide) <i>a</i> oxygenase (CAO) is required for Chl <i>b</i> synthesis. Conversion in vitro of Chlide <i>a</i> to Chlide <i>b</i> by purified recombinant CAO occurs at a low rate. In contrast, protochlorophyllide (Pchlide) <i>a</i> is converted rapidly and quantitatively to Chlide <i>b</i> by CAO in a membrane fraction from cells of <i>Chlamydomonas reinhardtii y-1</i> when incubated in the presence of polyaromatic <i>m</i>-phenanthroline. This is surprising because Pchlide <i>a per se</i> is not a substrate for CAO. A retrospective analysis of our previous data suggests that phenanthroline mimics Chlide <i>a</i> and pairs with Pchlide <i>a</i> to lower its redox potential, thereby activating Pchlide <i>a</i> as a substrate. A conserved tyrosine residue occurs as a stable radical in CAO and initiates the transformation of the 7-methyl group of Pchlide <i>a</i> to the 7-formyl group of Pchlide <i>b</i>. Replacing this tyrosine with alanine abolished CAO activity. We conclude that Pchlide <i>b</i> is synthesized from Pchlide <i>a</i> by an unusual, radical-mediated/based mechanism that directly involves molecular oxygen. In vivo,<i> s</i>ubsequent reduction of the C17-C18 double bond by a light-independent Pchlide reductase activity would be facilitated by the electronegative carbonyl of the 7-formyl group to generate Chlide <i>b</i>.</p>