The utility of peptide nucleic acid fluorescence in situ hybridization (PNA FISH) for the detection of Acinetobacter spp. and Pseudomonas aeruginosa was evaluated on broth suspensions and spiked blood cultures of ATCC strains and clinical isolates with select gram-negative rods. After testing 60 clinical isolates, PNA FISH had a sensitivity and specificity of 100 and 100 , respectively, for Acinetobacter spp. and 100 and 95 , respectively, for P. aeruginosa. PNA FISH was able to detect both pathogens simultaneously and directly from spiked blood cultures.