Micro-optical systems based on refractive microlenses are investigated. These systems are integrated on a chemical chip. They focus an excitation beam into the detection volume (microliter or even submicroliter scale) and collect the emitted light from fluorescent molecules. The fluorescence must be carefully separated by spatial and spectral filtering from the excitation. This paper presents the ray tracing simulation, fabrication, and measurement of three illumination systems. The measurements show that an adroit placement and combination of microfabricated lenses and stops can increase the separation between the excitation light and the fluorescence light. Moreover we present the successful detection of a 20 nM Cy5™ (Amersham Life Science Ltd.) solution in a 100-μm-wide and 50-μm-deep microchannel (excitation volume ≈250 pL) using one of these illumination systems. The microchemical chip with the micro-optical system has a thickness of less than 2 mm. © 2001 Society of Photo-Optical Instrumentation Engineers.