Recently sodium fluorescein has been used to determine viability of tissue and assess blood flow. No study, however, has documented the correlation of surface fluorescence as measured by the dermatofluorometer with the actual distribution of fluorescein in tissue. Abdominal skin flaps were raised on Sprague‐Dawley rats. Following intravenous injection of sodium fluorescein, skin surface fluorescence was measured and compared with tissue fluorescein concentration. This study confirms that skin surface fluorescence correlates well with actual fluorescein distribution in tissue. Furthermore, the study clearly illustrates the statistically significant variation of surface fluorescence as a function of distance from the vascular pedicle.