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In the present work a rapid and sensitive non-radioactive assay for the determination of cellular dimethylallyl diphosphate (DMADP) was developed and used for the analysis of the diurnal variation of DMADP levels in oak leaves. The method is based on the acid-catalysed hydrolysis of DMADP to isoprene, which subsequently is determined by gas chromatography. Diurnal variation of cellular DMADP levels in oak leaves of young saplings was measured on 6 days in 1998 and 1999, showing a 2 to 3-fold light-dependent increase from approximately 15 pmol mg−1 DW in the night to 31–75 pmol mg−1 DW around noon. The leaf DMADP contents showed a significant positive correlation with net assimilation and isoprene emission rates, indicating that the availability of cellular DMADP might be an important regulatory factor of leaf isoprene emission.