The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and retains a distinctive association with the microtubule cytoskeleton, comprises an elliptical patch of rhodopsin photoreceptors in the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast and serves as a model for understanding how organelles are formed and placed asymmetrically in the cell. This study describes the roles of several factors in the assembly and positioning of the eyespot. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. These proteins play interdependent roles: EYE2 and the ChR1 photoreceptor co-position in the absence of pigment granules, and the pigment granules are required to maintain the shape and integrity of the EYE2/ChR1 patch. The miniature-eyespot locus MIN2 affects eyespot size and likely regulates the amount of material available for eyespot assembly. The MLT2 locus regulates eyespot size, number, and asymmetry. A novel locus, PEY1, modulates the position of the eyespot on the anterior-posterior axis by affecting microtubule rootlet length. A working model is developed wherein rootlet microtubule-directed photoreceptor localization establishes connections in the chloroplast envelope with EYE2, which directs the site for pigment granule array assembly, and MLT2 is proposed to negatively regulate the levels of eyespot proteins.