Summary Non-radioactive hybridization methods were evaluated for the identification of microorganisms in mixed bioaerosols. A cultivation-dependent method, colony hybridization, was compared to a direct, cultivation-independent approach, whole cell hybridization with fluorescently labeled oligonucleotides. After sampling of the aerosols by filtration, special processing of filters (cells) preceded hybridization with fluorescently, digoxigenin- or enzyme-labeled oligonuculeotide probes. Group, genus, or species affiliation of collected cells was analyzed with rRNA-targeted probes. Using nucleic acid probes directed against the multiple cloning site, plasmid bearing Escherichia coli colonies could be differentiated from wild-type colonies. The microbial composition of aerosols ranging from less than one to greater than 10 9 cells/m 3 air could be analyzed with appropriate hybridization formats: whole cell hybridization was only applicable to dense aerosols, colony hybridization yielded best results with lower concentrations. After a short incubation period (several hours), a combination of both formats could be used to rapidly determine the fraction of culturable cells within a bioaerosol. When applying these techniques for the monitoring of aerosols generated by standard microbiological laboratory procedures, low concentrations of airborne Escherichia coli cells (1–450 m −3) could be detected. Compared to conventional air monitoring techniques, hybridization with nucleic acid probes should allow more rapid and reliable detection of airborne microorganisms including genetic engineered microorganisms.