ABSTRACTCyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (forGGDEFdomainprotein fromStaphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation inStaphylococcus epidermidis. Inactivation ofgdpSimpaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereasgdpSoverexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of theicaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevatingicaADBCmRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevateicaADBCexpression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented thegdpSmutation. Furthermore, heterologous diguanylate cyclases expressed intransfailed to complement thegdpSmutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. ThegdpSgene fromStaphylococcus aureusexhibited similar characteristics to itsS. epidermidisortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increasedicaADBCmRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.