SUMMARY The flight muscles of Drosophila are highly enriched with mitochondria; but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. We report the mechanism of CI biogenesis in Drosophila flight muscles; and show that it proceeds via the formation of ~315-, ~550-, and ~815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting an ~700 kDa transient CI assembly intermediate into the ~815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo; and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.