Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and β-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased α-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP α-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of β-amyloid peptides 1–40 or 1–42 as measured by ELISA, suggesting that only the α-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.