Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL, as a fluorescent substrate for HDACs. Now we present a modification of MAL called Z-MAL that is characterized by an increased rate of conversion by histone deacetylases of classes I and II and the recently discovered sirtuins (histone deacetylases class III). MAL and Z-MAL are the first nonradioactive substrates for class III enzymes. The new substrate Z-MAL allows for shorter assay times in inhibitor screening and is applicable to diverse sources of deacetylase activity even with completely different catalytic mechanisms. Interestingly, MAL shows some relative preference toward class II, indicating that subtype selectivity in small-molecule HDAC substrates might be obtained.