Amplified fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-specific digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and specifity of the tests were determined in titration studies with purified homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% specifity for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-amplified DNA and polymerase chain reaction (PCR) amplificates of 100 fg homologous template DNA. There was no reaction with heterologous strains when amplificates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for field samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and specificity, and high predictive values of positive or negative results. There was no significant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.