Expression of the Escherichia coli StpA protein was investigated and a functional comparison undertaken with the structurally analogous nucleoid protein H-NS. Analysis of stpA and hns expression indicated that although stpA transcript levels are much lower than those of hns, the two gene products are capable of both negative autogenous control and cross-regulation. Examination of cellular proteins in stpA, hns, or stpA-hns backgrounds revealed that StpA can repress and activate a subset of H-NS-regulated genes. Mechanistic parallels in regulation of gene expression are indicated by the ability of both proteins to inhibit transcription from promoters containing curved DNA sequences, and to form nucleoprotein structures that constrain DNA supercoils. Despite their functional similarities, each molecule is capable of independent activities. Thus, H-NS regulates a class of genes that are unaffected by StpA in vivo, whereas StpA has much stronger RNA chaperone activity in vitro. We therefore propose that in addition to its role as a molecular back-up of H-NS, StpA's superior effect on RNA may be exploited under some specific cellular conditions to promote differential gene expression.