Activation of members of the STAT (signal transducers and activators of transcription) family of latent transcription factors is an early event following the binding of many cytokines to their cognate receptors. Although the patterns of STATs activated by different cytokines are well described, the consequences of differential STAT activation are less well studied. We show by mutational analysis that STAT binding elements (SBEs) exist that discriminate between STAT complexes containing STAT1 alpha, STAT3 or both, and that these elements show altered cytokine responsiveness. We also show that in the context of a minimal promoter, single and multiple SBEs exhibit strikingly different patterns of transcriptional activation in response to IFN-gamma, IL-6, OSM or LIF. These differences in transcriptional activation are correlated with the differential ability of these cytokines to activate STAT1 alpha, STAT3 or both. Our results show that the pattern of STATs activated by a cytokine and the arrangement and sequence of the SBEs in the responding promoter have a profound effect on the ability of the cytokine to elicit a transcriptional response.