Translational regulation has been to shown to play major roles in the patterning of the early Drosophila embryo. The eIF4G family member NAT1/p97/DAP5 has been identified as a novel translational repressor. To genetically dissect the in vivo function of this unconventional eIF4G-related translational regulator, Drosophila NAT1 (dNAT1) mutants were isolated using a reverse-genetics approach. Four transposon insertion mutants and a deletion mutant affecting the dNAT1 locus were analyzed. Genetic complementation tests and germline rescue using a 12 kb dNAT1 genomic DNA fragment revealed these to be loss-of-function mutants. One P-element insertion line, dNAT1(GS1.), shows severe embryonic lethality and abnormal germband extension. Abnormalities at metamorphosis were also found, including defective head eversion and salivary gland degeneration in the hypomorphic allele dNAT(ex1). A phenotypic analysis of dNAT1 mutants suggests that dNAT protein plays a specific rather than general role in translational regulation.