Fluorescence diagnostics based on aminolaevulinic acid (ALA) fluorescence has been suggested as an in vivo pre-surgical tool for tumour demarcation. We performed fluorescence diagnostics of 35 basal cell carcinomas (BCCs) undergoing photodynamic therapy (PDT) using methyl-aminolaevulinate (MAL). In addition, a semi-automated thresholding algorithm was implemented to detect the potential tumour region. The mean tumour fluorescence contrast was found to be 1.65 ± 0.06 during the first MAL-PDT session, and increased to 1.84 ± 0.07 at the second treatment (p < 0.01). This could imply that disruption of the skin barrier and inflammatory responses after the first session of PDT led to higher accumulation of proto-porphyrin IX during the second session of PDT. The tumour areas detected based on fluorescence in small BCCs (< 1 cm(2)) were in general (n = 18/23) larger than the visual clinical tumour size. In addition, the fluorescence contrast using MAL (1.65 ± 0.06) was found to be significantly higher (p<10(-4)) than the contrast (data from previous study) after application of ALA (1.20 ± 0.06). Thus, MAL generally provides higher tumour contrast than ALA in BCCs, and should be preferred for use in fluorescence diagnostics. Correlation between fluorescence, lack of treatment response and/or pain was not observed.