This article discusses muscle pain concepts in the context of myofascial pain syndrome (MPS) and summarizes microdialysis studies that have surveyed the biochemical basis of this musculoskeletal pain condition. Though MPS is a common type of non-articular pain, its pathophysiology is only beginning to be understood due to its enormous complexity. MPS is characterized by the presence of myofascial trigger points (MTrPs), which are defined as hyperirritable nodules located within a taut band of skeletal muscle. MTrPs may be active (spontaneously painful and symptomatic) or latent (non-spontaneously painful). Painful MTrPs activate muscle nociceptors that, upon sustained noxious stimulation, initiate motor and sensory changes in the peripheral and central nervous systems. This process is called sensitization. In order to investigate the peripheral factors that influence the sensitization process, a microdialysis technique was developed to quantitatively measure the biochemical milieu of skeletal muscle. Biochemical differences were found between active and latent MTrPs, as well as in comparison with healthy muscle tissue. In this paper we relate the findings of elevated levels of sensitizing substances within painful muscle to the current theoretical framework of muscle pain and MTrP development.