Mice lacking transcription factor AP-2α exhibit defects in the formation of the head, body wall, heart, neural tube, eye, and limbs, reflecting important sites of AP-2α expression in the developing embryo. AP-2α is also expressed in the postnatal mammary gland and has been linked to tumor progression and defects in growth regulation in the breast. We have used a transgenic mouse approach to identify tissue-specific cis-acting sequences associated with expression of the human AP-2α gene. Our analysis indicates that multiple elements located throughout the gene contribute to expression in the trigeminal ganglia, spinal cord, mammary gland, and epidermis. A discrete cis-element located within the fifth intron is required for expression in the face and limbs, and we have derived a permanent line of AP-2α::lacZ transgenic mice to assess expression of this latter enhancer throughout morphogenesis. We also introduced this transgene into an AP-2α–null mouse background and detected subtle alterations of its expression within the progress zone and apical ectodermal ridge of the forelimbs. Similar changes in lacZ expression were observed within the zeugopod, and these correlated with defects in radius condensation in AP-2α–knockout mice. Taken together, these findings indicate that cell:cell communication within the forelimb is altered in the absence of AP-2α and reveal novel regulatory potential for AP-2α in limb development. Developmental Dynamics 228:194–207, 2003. © 2003 Wiley-Liss, Inc.