Light is necessary for hydrogenation of the D ring of protochlorophyllide leading to chlorophyllide formation in higher plants (light-dependent pathway), but it is not essential in phototrophic bacteria (dark pathway). Both pathways, however, occur in some algae, mosses, ferns, and gymnosperms, and each chloroplast genome of these organisms contains three genes, chlL, chlN, and chlB, encoding the three subunits of light-independent protochlorophyllide reductase (LIPOR) required for protochlorophyllide reduction in the dark. In this study, the three LIPOR genes chlL, chlN, and chlB were cloned from the chloroplast of Chlorella protothecoides CS-41 (CSIRO), which grew heterotrophically with considerable chlorophyll yield. Phylogenetic analysis of ChlL/BchL showed that C. protothecoides CS-41 and Chlorella vulgaris C-27 were closely related. Alignment of their amino acid sequences demonstrated that the conserved domains, including the ATP-binding motif and the Fe-S binding motif in the three subunits, were similar to those in nitrogenases. The three-dimensional structural model of ChlL revealed a hypothetical Fe-S center for redox control. Results from RT-PCR amplification indicated that the chlL gene in C. protothecoides contained a 951-bp intron, and the splicing catalytic core structure was similar to that of the light-regulated intron in the psbA gene of Chlamydomonas. The three genes were expressed in E. coli BL21. The sizes of ChlL, ChlN, and ChlB were estimated to be 38, 49, and 58 kDa, respectively, based on the SDS-PAGE analysis.