Pollen exine, mainly composed of sporopollenin,plays important roles during microspore development. It hasbeen reported that Acyl-CoA Synthetase5 (ACOS5) is requiredfor sporopollenin biosynthesis in Arabidopsis. Here we showthat ACOS5 is essential for primexine formation duringArabidopsis microspore development. Through genetic screen,we identified a point mutation of ACOS5 allele, acos5-2,showing abnormal microspore development. Its microsporeswere degenerated and aborted after released from the tetrads. Transmission electron microscopy showed that primexineformation was reduced in acos5-2 mutant as compared tothat of the wild-type. Consequently, sporopollenin wasaggregated and randomly deposited on the microspores. Insitu hybridization indicated that the key regulators of tapetumdevelopment, DYT1 and TDF1, are required for the expressionof ACOS5 in tapetum. Furthermore, the GUS reporter showedthat the 593-bp promoter sequence was sufficient for theexpression of ACOS5 in the anther. Our data provide evidencethat ACOS5 is required for primexine formation andsporopollenin deposition during microspore development.