Background: Unexpected phenotypes resulting from morpholino-mediated translational knockdown of Pitx3 in Xenopus laevis required further investigation regarding the genetic networks in which the gene might play a role. Microarray analysis was, therefore, used to assess global transcriptional changes downstream of Pitx3. Results: From the large data set generated, selected candidate genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Conclusions: We have identified four genes as likely direct targets of Pitx3 action: Pax6, β Crystallin-b1 (Crybb1), Hes7.1, and Hes4. Four others show equivocal promise worthy of consideration: Vent2, and Ripply2 (aka Ledgerline or Stripy), eFGF and RXRα. We also describe the expression pattern of additional and novel genes that are Pitx3-sensitive but that are unlikely to be direct targets. Developmental Dynamics 241:1487–1505, 2012. © 2012 Wiley Periodicals, Inc.