A microarray was constructed and applied to transcriptional profiling and genetic variation studies of Mycoplasma hyopneumoniae. The genome sequence enabled the construction of the microarray to allow a global approach to understanding fundamental processes in M. hyopneumoniae . These studies focused on whether M. hyopneumoniae regulates its genes under different environmental conditions and if genetic changes can be correlated with virulence. The microarray consisted of 632 open reading frames represented by polymerase chain reaction products and were used in a two-color experimental design. Data were analyzed using a mixed linear statistical model. Unique features implemented in these studies included the printing of two complete arrays per substrate, reducing the slide to slide variation; the scanning of each dye channel of each array at different laser power settings to increase the dynamic range of expression measurement; and the use of a unique set of hexamer primers to generate fluorescently labeled targets for microarray analysis. The first series of studies focused on transcriptional profiling during heat shock and iron deprivation, two environmental changes that M. hyopneumoniae encounters on the respiratory epithelial surface during disease. Key genes responsive to these stresses were identified, and interestingly, fifty-three were regulated in common to the two conditions at p<0.05. Since adherence to swine cilia is a prerequisite for colonization and disease, the next series of studies involved the comparison of an adherent, pathogenic strain 232 to a nonpathogenic, nonadherent type strain J. In conjunction with this study, two strain 232 adherence variants, one high and one low, were also compared. Thirty genes were identified that differed between the strains 232 and J. Nineteen genes were up-regulated in the high adherent variant including the heat shock protein DnaJ, and fifteen genes were up-regulated in the low adherent strain. In a comparison of eight field isolates to strain 232, two were indistinguishable from strain 232 and the others varied in an many as twenty-five loci and as few as one. In summary, these studies attempt to unravel some of the underlying mechanisms and pathways M. hyopneumoniae uses to infect the host and cause disease.