OBJECTIVE To investigate the reproducibility of direct and axon-reflex vasodilator responses to increasing doses of acetylcholine administered transcutaneously by iontophoresis. METHODS Increasing doses of acetylcholine ions were introduced into the skin of the volar forearm with weak electric currents. The elicited vasodilatation was measured with laser Doppler flowmetry at the direct site of acetylcholine administration and approximately 5 mm from this site (within the distribution of axon-reflex vasodilatation). To investigate the reproducibility of these measures, iontophoresis was carried out twice at one site after a 50 min interval and once at another site on the forearm of 16 healthy females. Raw scores and data expressed as percentages of baseline, maximum, and response to heating were analysed to determine the most reliable form. RESULTS The dose-response curve was significantly greater at the upper site on the forearm than at the lower site for direct vasodilator responses, but did not differ between the upper and lower forearm for axon-reflex responses. The various forms of data expression yielded weak to moderate correlations for vasodilatation between sites, and moderate to strong correlations when the iontophoresis was repeated at the same site. Direct responses were most reproducible when expressed in proportion to levels recorded after 5-6 min of local 42 degrees C heating (percentage of shared variance was 54.6% within sites and 61.0% between sites). CONCLUSIONS Cutaneous vasodilatation to acetylcholine iontophoresis is reproducible over time at a single site but varies across sites. This will allow future research to assess microvascular function before and after a laboratory manipulation in a single session.