Polymorphisms within the gene encoding macrophage migration inhibitory factor (MIF) have been associated with susceptibility to inflammatory diseases such as rheumatoid arthritis and increased risk of developing sepsis. We investigated the effects of the MIF-173G > C polymorphism and the MIF-794 CATT microsatellite on MIF expression. These are in moderate linkage disequilibrium. Mononuclear cells from healthy donors were stimulated with bacterial pathogens associated with sepsis (Streptococcus pneumoniae or Escherichia coli). MIF mRNA and protein levels were measured by real-time polymerase chain reaction and ELISA, respectively. Carriage of the C allele of MIF-173G > C or the 7 CATT repeat of the MIF-794 microsatellite correlated with lower basal and stimulated MIF mRNA levels. However, levels of intracellular and extracellular MIF protein were similar. This discordance between effects on MIF mRNA and protein was not explained by differential effects of genotype on stability of MIF mRNA (detected by actinomycin D mRNA chase). Gel shift assays revealed no differences in the profile of nuclear proteins from mononuclear cells bound by the G and C alleles of MIF-173G > C, but alleles at the microsatellite marker showed differential binding. Our data suggest that the MIF-794 CATT microsatellite influences transcription by differential binding of nuclear transcription factors. This may impact on inflammatory processes.