Background and Objectives Compared to the conventional management of cervical intraepithelial neoplasia (CIN) the potential advantage of photodynamic therapy (PDT) for the treatment of cervical human papilloma virus (HPV)-related disease encompasses a minimal invasive procedure with reduced risk of profuse bleeding as a consequence of conization, and possibly more favorable long-term results avoiding cervical stenosis. At present little is known about the precise time-dependent distribution and histological localization of hexaminolaevulinate (HAL) induced protoporphyrin IX (PPIX) fluorescence in healthy tissue and in CIN. The aim of this study was to use ex vivo fluorescence microscopy to determine whether PPIX is selectively induced by neoplastic cells of the cervical epithelium at various times after topical application. Study Design/Materials and Methods Cold cream containing 0.5% HAL was applied by means of cervical cap over various periods of time. We analyzed 52 healthy cervical mucosa and 84 CINs. Results At time delay 100 (±10) minutes, high epithelial fluorescence and a significant selectivity between epithelium and underlying lamina propria was found. By contrast, no significant difference between healthy and neoplastic tissues, or between low and high-grade epithelial dysplasia (P 0.05), was observed at any time point. Conclusions Application of HAL 0.5% cream to the cervix induced selective fluorescence in epithelial cells. The optimal ratio with a homogeneous PPIX distribution was obtained after 100 ( ± 10) minutes cream application, which should be evaluated further for PDT.