Direct intratumoural (IT) administration of adenovirus is widely used, however little is known about the resulting distribution of virus particles. Here we have evaluated the influence of tumour size, volume of injectate and occlusion of injection sites (to prevent retrograde seepage) on particle biodistribution and transgene expression. In subcutaneous MDA-231 xenografts, IT injection of relatively large volumes (4 x 20% (vol/vol) injections) resulted in just 40% of the administered dose being retained in tumour tissue after 30 min, with 15% in the liver thought to reflect systemic 'overflow'. Occlusion of the injection sites using surgical adhesive increased retention of the vector to 80% in the tumour with no increase in liver levels. Spread of expression was enhanced using multiple injection sites, but not by using larger injectate volumes. In ZR75.1 breast carcinoma xenografts virus distribution was different, with no evidence of systemic overflow leading to hepatic transduction following IT injection. Typically, clinical doses employ up to 30% vol/vol IT injections. Depending on the tumour, this may give considerable systemic overflow and might account for the high frequency of fevers observed. Virus performance might be improved by tailoring volumes and frequency of IT injection for tumour biology or histotype.