Spectrin α2 (αII-spectrin) is a scaffolding protein encoded by the Spna2 gene and constitutively expressed in most tissues. Exon trapping of Spna2 in C57BL/6 mice allowed targeted disruption of αII-spectrin. Heterozygous animals displayed no phenotype by 2 years of age. Homozygous deletion of Spna2 was embryonic lethal at embryonic day 12.5 to 16.5 with retarded intrauterine growth, and craniofacial, neural tube and cardiac anomalies. The loss of αII-spectrin did not alter the levels of αI- or βI-spectrin, or the transcriptional levels of any β-spectrin or any ankyrin, but secondarily reduced by about 80% the steady state protein levels of βII- and βIII-spectrin. Residual βII- and βIII-spectrin and ankyrins B and G were concentrated at the apical membrane of bronchial and renal epithelial cells, without impacting cell morphology. Neuroepithelial cells in the developing brain were more concentrated and more proliferative in the ventricular zone than normal; axon formation was also impaired. Embryonic fibroblasts cultured on fibronectin from E14.5 (Spna2−/−) animals displayed impaired growth and spreading, a spiky morphology, and sparse lamellipodia without cortical actin. These data indicate that the spectrin–ankyrin scaffold is crucial in vertebrates for cell spreading, tissue patterning and organ development, particularly in the developing brain and heart, but is not required for cell viability.