Background: The bacterium Staphylococcus aureus causes significant morbidity and mortality in humans, primarily due to the emergence of strains that are resistant to antibiotics notably methicillin-resistant S. aureus (MRSA) isolates. Development of effective strategies for the control and treatment of MRSA infections may best be achieved through 'omics' approaches, which first requires cloning the entire set of S. aureus' protein-encoding open reading frames (ORFs), or ORFeome. Results: The complete genome sequence of S. aureus strain Mu50 has 2697 predicted protein-coding ORFs. Based on the sequence of this strain we designed PCR primers to construct from an S. aureus (non-MRSA) clinical isolate an ORFeome library that contains 2562 unique Gateway® entry clones (95% coverage), each corresponding to a defined ORF. The high quality of the ORFeome library was verified by DNA sequencing and PCR amplification, and its functionality was demonstrated by expressing recombinant proteins and observing protein interactions in a yeast 2-hybrid homodimerization screen. Conclusion: This first ORFeome library for S. aureus provides an essential new tool for investigating the systems biology of this important pathogen.