Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes， respectively， and are devoid of any polyamine when grown in polyamine-free media. In contrast to bacteria such as Pseudomonas aeruginosa， Campylobacter jejuni and Agrobacterium tumefaciens， which absolutely require polyamines for growth， S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore， S. aureus and E. faecalis form biofilms normally without polyamines， and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines， including norspermidine， eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N-acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine， spermine and norspermidine， that spermine is the more preferred substrate， and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy， polyamine-independent growth and biofilm formation， and presence of functional polyamine N-acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology. This article is protected by copyright. All rights reserved.