Results of in vitro experiments have suggested the existence of at least three pathways by which nuclear-encoded proteins are targeted to the chloroplast thylakoid membrane. However, few components of the targeting machinery have been identified and the relationship between the three pathways is not clear. To investigate mechanisms underlying thylakoid protein targeting, we identified nuclear mutations in maize that cause targeting defects. We found two mutations, tha1 and hcf106, that disrupt the localization of different sets of proteins to the thylakoid lumen. The tha1 mutation interferes with the targeting of one chloroplast-encoded protein, cytochrome f, and three nuclear-encoded proteins, plastocyanin, the psaF gene product and the 33 kDa subunit of the oxygen-evolving complex. The hcf106 mutation interferes with the targeting of the 16 and 23 kDa subunits of the oxygen-evolving complex. The tha1 and hcf106 phenotypes provide the first in vivo evidence supporting the existence of two distinct thylakoid-targeting pathways. Their phenotypes also provide evidence that one chloroplast-encoded protein, cytochrome f, engages the 'tha1' pathway, indicating that nuclear- and chloroplast-encoded proteins can be targeted via common machinery.