Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168. The S. epidermidis tagF gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into B. subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned S. epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike B. subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S. epidermidis is not linked to any other tag genes. We attempted to disrupt the chromosomal tagF gene in S. epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5' end of tagF on a similarly sized DNA fragment was able to integrate. This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.