Hfq is an RNA-binding protein that participates in the regulatory activity of small non-coding RNAs (sRNAs) in many species of bacteria. Hfq protein was first crystallized from Staphylococcus aureus and this crystal structure constitutes a hallmark for bacterial Sm-like proteins. Paradoxically, however, the functional relevance/role of S. aureus Hfq (Hfq(SA)) remains uncertain, as growing evidence suggests that the hfq(SA) gene is expressed at very low levels or unexpressed in many S. aureus strains. To gather further insight, in the present work we exchanged the structural portion of the hfq gene of Salmonella enterica serovar Typhimurium (hfq(STM)) with hfq(SA) and analyzed the effects of the replacement on various Hfq-related phenotypes. Our results show that the replacement strain - in spite of expressing Hfq(SA) at levels comparable to Hfq(STM) in wild-type Salmonella - behaves as an hfq null mutant in three discrete small RNA-mediated regulatory responses. These defects correlate with an abrupt reduction in the intracellular concentration of sRNAs, as observed in an hfq null mutant. Failure of Hfq(SA) to protect Salmonella sRNAs from degradation suggests that Hfq(Sa) does not bind to these sRNAs. A parallel study with the Borrelia burgdorferi hfq gene (hfq(BB)) gave essentially identical results: when made from a single copy chromosomal gene, Hfq(BB) fails to substitute for Hfq(STM) in sRNA-mediated regulation.