Cutaneous squamous (pre)malignancies can be classified according to the keratinocytic intraepidermal neoplasia (KIN) classification. Aneuploidy can be seen as the result of chromosomal aberrations leading to altered DNA content and has been strongly associated with malignancy. Hyperproliferation is also strongly associated with tumorigenesis. The aim of the study was to analyse the presence and the amount of aneuploidy and proliferation in the progression from intraepithelial neoplasm to microinvasive carcinoma (miSCC). For this purpose, nuclei were isolated from 116 formalin-fixed KIN lesions from 68 patients in which DNA content was measured by flow cytometry. Proliferation was assessed by immunohistochemical staining for Ki67 as well as by flow cytometry. Aneuploidy was increasingly found in higher KIN lesions, but not in normal skin. However, in miSCC aneuploidy was relatively less frequently found. DNA indices (mean +/- SE) of KIN III-lesions (1.57 +/- 0.05) were significantly lower compared with KIN I/II lesions (1.71 +/- 0.05). Ki67 expression was strongly positively correlated with KIN grade, and proved to be a good adjunct in the classification of KINs. Thus, aneuploidy occurred more frequently in higher KIN lesions, indicating cumulative damage during KIN progression. The lower frequency of aneuploidy in miSCC compared with KIN III may point at alternative routes towards invasive carcinoma besides serial progression through all three KIN stages. Ki67 expression appears a valuable marker in the classification of KINs.