The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.