Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.