Behavioural and hereditary conditions are known to decrease mitochondrial volume and function within skeletal muscle. This reduces endurance performance, and is manifest both at high- and low-intensity levels of exertion. A programme of regular endurance exercise, undertaken over a number of weeks, produces significant adaptations within skeletal muscle such that noticeable improvements in oxidative capacity are evident, and the related decline in endurance performance can be attenuated. Notwithstanding the important implications that this has for the highly trained endurance athlete, an improvement in mitochondrial volume and function through regular physical activity also endows the previously sedentary and/or aging population with an improved quality of life, and a greater functional independence. An understanding of the molecular and cellular mechanisms that govern the increases in mitochondrial volume with repeated bouts of exercise can provide insights into possible therapeutic interventions to care for those with mitochondrially-based diseases, and those unable to withstand regular physical activity. This review focuses on the recent developments in the molecular aspects of mitochondrial biogenesis in chronically exercising muscle. Specifically, we discuss the initial signalling events triggered by muscle contraction, the activation of transcription factors involved in both nuclear and mitochondrial DNA transcription, as well as the post-translational import mechanisms required for mitochondrial biogenesis. We consider the importance and relevance of chronic physical activity in the induction of mitochondrial biogenesis, with particular emphasis on how an endurance training programme could positively affect the age-related decline in mitochondrial content and delay the progression of age- and physical inactivity-related diseases.