In this study we performed in situ hybridization using biotin-labelled total genomic DNA of Haemophilus influenzae type b as a probe on: (1) smears containing bacteria cultured in vitro: all haemophilus species that can be found in the human respiratory tract appeared to be positive and a large number of other bacterial species appeared to be negative in this in situ hybridization test; (2) sputum smears from 287 patients with bronchitis: the hybridization test was positive on all but 2 of the 44 smears derived from patients whose culture yielded haemophilus and additionally on 12 smears derived from patients, whose culture was negative; and (3) sputum smears from 7 patients suffering from cystic fibrosis (CF): the hybridization test was positive in all these 7 sputum smears, while the culture only yielded haemophilus in 3 cases. The higher sensitivity of the hybridization test compared to culturing could mainly be explained by the failure to detect haemophilus in culture caused by masking due to overgrowth by other bacteria. In conclusion the in situ hybridization test, which can be performed in only 4 h, is a sensitive and specific method for the detection of haemophilus in sputum and is particularly useful in CF patients, where overgrowth by pseudomonas often interferes with diagnosis by culturing.