Converging evidence supports a role of serotonin (5-hydroxytryptamine; 5-HT) in barrel cortex development. Systemic administration of 5-HT-depleting drugs reduces cross-sectional whisker barrel areas in the somatosensory cortex (SSC) of neonatal rats. Here we assess the relative impact on barrel pattern formation of (i) 5-HT depletion and (ii) decreased brain growth, which is often associated with pharmacological 5-HT depletion, by comparing the effects of 5-HT-depleting drugs with those of reduced protein intake. Left hemisphere 5-HT levels in the SSC and right hemisphere whisker barrel areas were assessed at postnatal day 6 (P6) in the same animal following injection of p-chloroamphetamine (PCA) or p-chlorophenylalanine (PCPA) at P0. Both drugs significantly reduced cortical 5-HT content and mean barrel areas at P6, but also body and brain growth. Differences in brain weight accounted for 84.4% of the variance in barrel size, with negligible contributions by cortical 5-HT content. PCPA-treated animals sacrificed at P14 yielded similar trends, albeit less pronounced. Finally, reduced protein intake resulted in lower body weight and cortical 5-HT levels at P6, but yielded no change in brain weight or mean barrel area. Barrel formation therefore appears markedly less sensitive to 5-HT depletion per se than to drug-induced growth impairment.