In a striking case of evolutionary convergence, polarized cell layers with ring-like diffusion barriers have evolved in both plant and animal lineages independently. In plants, ring-like Casparian strips become localized by the CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs). The mechanism of this striking localization, however, has remained enigmatic. Here we present a genetic screen aimed at isolating determinants of CASP localization. One of the mutants, lord of the rings 2 (lotr2)/exo70a1, displays dramatic de-localization of CASPs into randomly localized microdomains. EXO70A1 is a subunit of the exocyst complex, a central component of secretion in eukaryotes. Irradiation of EXO70 subunit genes in plants has suggested specialization of this conserved complex. Intriguingly, lotr2/exo70a1 does neither affect secretion of the CASPs, nor that of other membrane proteins in the endodermis, thus separating exocyst activity in localization from a general defect in secretion. Our results establish EXO70A1 as a central player in Casparian strip formation, generating a transient positional information that will be translated into a precisely localized cell wall modification.