The reagents Lumigen PS-1 and Lumigen PS-3 were originally developed as chemiluminescent substrates for ultrasensitive detection of horseradish peroxidase (HRP) in homogeneous solution and membrane blotting assays. However, an additional unique feature of these acridan-based reagents is the generation of a fluorescent species on reaction with peroxidase, a property which has been termed as chemifluorescence. These reagents, therefore, represent the first dual-use substrates enabling both chemiluminescent and fluorescent detection. We have developed several additional acridan-based substrates for fluorescent detection of HRP which are capable of subattomole detection sensitivity. By varying several structural parameters within the class of compounds we have produced substrates which either produce fluorescence alone or both chemiluminescence and fluorescence.