rRNA-based studies, to assess microbial communities, rely on the accurate amplification of the corresponding genes from the original DNA sample. Here we present an analysis and re-evaluation of commonly used primers for amplifying DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the rRNA of Escherichia coli). We propose a forward primer formula (27f) that includes three sequences that are not commonly found. We compare our proposed formula with two common alternatives using linear amplification—providing a reverse primer-independent assessment—and in combination with the reverse primer 1492 (1492r) under appropriate PCR conditions for generating community rRNA gene clone libraries. For analyses of DNA from human vaginal samples,