GAP-43 has been implicated in axonal pathfinding and sprouting, synaptic plasticity, and neurotransmitter release. However, its effect on cortical development in vivo is poorly understood. We have previously shown that GAP-43 knockout (−/−) mice fail to develop whisker-related barrels or an ordered whisker map in the cortex. Here we used cytochrome oxidase (CO) histochemistry to demonstrate that GAP-43 heterozygous (+/−) mice develop larger than normal barrels at postnatal day 7 (P7), despite normal body and brain weight. Using serotonin transporter (5HT-T) histochemistry to label thalamocortical afferents (TCAs), we found no obvious abnormalities in other somatosensory areas or primary visual cortex of GAP-43 (+/−) mice. However, TCA projections to (+/−) primary auditory cortex were not as clearly defined. To clarify the mechanism underlying the large-barrel phenotype, we used lipophilic (DiI) axon labeling. We found evidence for multiple pathfinding abnormalities among GAP-43 (+/−) TCAs. These axons show increased fasciculation within the internal capsule, as well as abnormal turning and branching in the subcortical white matter. These pathfinding errors most likely reflect failures of signal recognition and/or transduction by ingrowing TCAs. In addition, many DiI-labeled (+/−) TCAs exhibit widespread, sparsely branched terminal arbors in layer IV, reflecting the large-barrel phenotype. They also resemble those found in rat barrel cortex deprived of whisker inputs from birth, suggesting a failure of activity-dependent synaptogenesis and/or synaptic stabilization in (+/−) cortex. Our findings suggest that reduced GAP-43 expression can alter the fine-tuning of a cortical map through a combination of pathfinding and synaptic plasticity mechanisms. J. Comp. Neurol. 462:252–264, 2003. © 2003 Wiley-Liss, Inc.