Our goal has been to study the distribution and effects following a single intramuscular injection of a substance using India ink as a tracer. We injected 30 microl India ink in the gastrocnemius muscle group of C57Bl10 mice. Hematoxylin-Eosin, Trichrome stains and polyclonal anti-laminin, anti-collagen-IV and anti-dystrophin were used. The liquid spreads in all directions mainly following the perimysial and epimysial septae. The collagen bundles act as physical barriers preventing passage of the ink particles. In the area of the injection site, necrosis of the fibres is associated with disruption of the basement membrane. In the zones adjacent and distal to the injection site, the liquid progresses by pushing the muscle fibres apart with preservation of the basement membrane. Research based on intramuscular injection of substances should take the following into consideration: a) anatomy of the muscle group injected, b) routes of distribution of the substance, c) types of lesions produced with relation to the site of injection of the liquid, and d) size of the particles of the injected substance.