MCu(II)-diacetyl-bis(A-methyl-thiosemicarbazone) (64Cu-ATSM) has been proposed as a positron emitting radiotracer to delineate tumour hypoxia using positron emission tomography (PET). In this study evaluation of this radiotracer was carried out in the P22 carcinosarcoma BD-9 rat tumour model. The distribution of 64Cu-ATSM with the immunohistochemical marker of hypoxia, pimonidazole, was compared using autoradiography and pimonidazole staining at early (1 hour) and late (20.5 hours) time-points after tracer injection however no correlation was found. The impact of tumour blood flow in determining tumour distribution of 64Cu-ATSM Gadolinium- enhanced MRI scanning was investigated. This showed a correlation between 64Cu- ATSM and blood flow up to twenty minutes after the administration of 64Cu-ATSM, an association beyond this time-point was not seen. A further study examined the effect of increased tumour hypoxia on uptake and retention of 64Cu-ATSM using the vascular disrupting agent combretastatin A4 phosphate (CA4P). No correlation between 64Cu-ATSM uptake and retention and hypoxia was seen in the tumours administered 64Cu-ATSM after CA4P administration, however a negative correlation between 64Cu-ATSM uptake and hypoxia was seen in the tumours receiving 64Cu-ATSM 10 minutes prior to CA4P administration. This strong negative association is likely to reflect the distribution of 64Cu-ATSM fixed at the time of administration of the vascular disrupting agent CA4P preventing its movement elsewhere in the tumour. In this tumour model 64Cu-ATSM distribution reflects tumour blood flow for twenty minutes after its administration. As such, fixation of the distribution of 64Cu-ATSM 10 minutes post-injection is likely to reflect tumour blood flow, explaining the negative association between 64Cu-ATSM and hypoxia observed in this part of the study. Evaluation of 64Cu-ATSM in the P22 tumour model has demonstrated that although it is tumour-avid it does not represent tumour hypoxia.