The spatial heterogeneity of the oxygen tension (pO2) in human and experimental tumours has been studied extensively, whereas studies of the temporal heterogeneity in pO2 are sparse. In the work reported here, pO2 was measured continuously over periods of at least 60 min in A-07 human melanoma xenografts by using the OxyLite fibre-optic oxygen-sensing device. The main purpose of the work was to establish the usefulness of the OxyLite system in measuring the temporal heterogeneity in pO2 in tissues and to characterise the fluctuations in tissue pO2 in A-07 tumours. The OxyLite device was found to be suitable for studies of the temporal heterogeneity in pO2 in tumours. However, potential pitfalls were identified, and reliable pO2 measurements require that precautions are taken to avoid these pitfalls, that is, erroneous pO2 readings caused by tissue trauma induced by the probe, probe movements induced by reflex actions of the host mouse and occasional probe drift. Significant fluctuations in pO2 were detected in the majority of the 70 tumour regions subjected to measurement. The fluctuations in different regions of the same tumour were in general temporally independent, implying that they were caused primarily by redistribution of the tumour perfusion rather than fluctuations in global perfusion. Fourier analysis of the pO2 traces showed that the pO2 usually fluctuated at frequencies lower than 1.5–2.0 mHz, corresponding to less than 0.1 cycle min−1. Haemodynamic effects may cause pO2 fluctuations in this frequency range, and hence, the redistribution of the perfusion could have been caused by morphological abnormalities of the tumour microvasculature. Moreover, acute hypoxia, that is, pO2 fluctuations around 10 or 5 mmHg, was detected in 20 of 70 regions, that is, 29% (10 mmHg), or 27 of 70 regions, that is, 39% (5 mmHg). The median fraction of the time these regions were acutely hypoxic was 73% (10 mmHg) or 53% (5 mmHg). Consequently, if A-07 tumours are adequate models of tumours in man, acute hypoxia may be a commonly occurring phenomenon in neoplastic tissues, and hence, acute hypoxia is likely to cause resistance to radiation therapy and promote tumour aggressiveness.