Trans-tracheal aspirations from 56 apparently healthy calves and 34 calves with clinical signs of pneumonia were collected in six different herds during September and November 2002. The 90 samples were cultivated and investigated by PCR tests targeting the species Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Mycoplasma dispar, and Mycoplasma bovirhinis. A PCR test amplifying the lktC-artJ intergenic region was evaluated and shown to be specific for the two species M. haemolytica and Mannheimia glucosida. All 90 aspirations were also analyzed for bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus, and bovine corona virus by antigen ELISA. Surprisingly, 63% of the apparently healthy calves harbored potentially pathogenic bacteria in the lower respiratory tract, 60% of these samples contained either pure cultures or many pathogenic bacteria in mixed culture. Among diseased calves, all samples showed growth of pathogenic bacteria in the lower respiratory tract. All of these were classified as pure culture or many pathogenic bacteria in mixed culture. A higher percentage of the samples were positive for all bacterial species in the group of diseased animals compared to the clinically healthy animals, however this difference was only significant for M. dispar and M. bovirhinis. M. bovis was not detected in any of the samples. BRSV was detected in diseased calves in two herds but not in the clinically healthy animals. Among the diseased calves in these two herds a significant increase in haptoglobin and serum amyloid A levels was observed compared to the healthy calves. The results indicate that haptoglobin might be the best choice for detecting disease under field conditions. For H. somni and M. haemolytica, a higher percentage of the samples were found positive by PCR than by cultivation, whereas the opposite result was found for P. multocida. Detection of P. multocida by PCR or cultivation was found to be significantly associated with the disease status of the calves. For H. somni a similar association with disease status was only observed for cultivation and not for PCR.