Second-generation nuclear medicine markers of tumour hypoxia have been synthesised and screened for hypoxic marking activity in cell cultures and in mouse tumours (EMT-6). Markers of the iodinated azomycin nucleoside class with greater water solubility and faster plasma clearance rates relative to iodoazomycin arabinoside (IAZA) were of particular interest. The test systems used to characterise hypoxic marking activity of compounds included (1) covalent linkage of radiolabelled markers to cells in suspension culture equilibrated with specific O2 concentrations; (2) biodistribution of radiolabelled markers in EMT-6 tumour-bearing mice; and (3) biodistribution in R3327-AT tumour-bearing rats by nuclear medicine procedures. Of the iodinated azomycin nucleosides produced to date, beta-D-iodoazomycin galactoside (beta-D-IAZG) and beta-D-iodoazomycin xylopyranoside (beta-D-IAZXP) exhibited high metabolism-dependent hypoxic cell uptake, rapid clearance kinetics from the blood and excellent tumour marking activity in vivo. Tumour-blood (T/B) ratio (a measure of tumour hypoxic fraction) was dependent upon EMT-6 tumour size and implantation site. The radioresistance of individual tumours was measured by in vivo/in vitro assay and correlated well with the T/B ratio of hypoxic marker. These studies have identified beta-D-IAZG and beta-D-IAZXP as effective hypoxic markers for planar and single photon emission computerised tomography (SPECT) imaging studies of tumour oxygenation.