The purpose of this study was to investigate where the pain comes from in chronic mid-portion Achilles tendinosis, by using ultrasonography and colour Doppler, immunohistochemistry, and diagnostic injections. Twenty-five tendons in 24 patients (mean age 47 years) with the clinical diagnosis of painful chronic mid-portion Achilles tendinosis, and 20 tendons in 14 controls (mean age 48 years) with pain-free Achilles tendons were examined with ultrasonography and colour Doppler. For the 25 painful tendons, a local anaesthetic was injected in the area with neovascularisation outside the ventral part of the tendon. Biopsies taken from tendinosis tissue in six patients were used for PGP 9.5 immunohistochemistry. A neovascularisation, inside and outside the ventral part of the area with tendon changes, was demonstrated in all painful tendinosis tendons, but not in any of the pain-free control tendons. After injection of the local anaesthetic towards the neovessels outside the ventral part of the tendon, the pain during tendon-loading activity was temporarily cured in all patients. The mean VAS-score for heel-raises decreased significantly from 75 mm to 6 mm. In biopsies, PGP 9.5 immunohistochemistry showed nerve structures in the vicinity of blood vessels. In conclusion, we demonstrate findings which support neovessels and accompanying nerves being the possible source of pain in chronic mid-portion Achilles tendinosis.