Twice-daily topical application of 5-fluorouracil 5% cream has been widely used to effectively treat actinic keratoses (AKs). In this study, once-daily application of 5-fluorouracil 5% cream for 4 weeks was evaluated in 12 patients, and monitoring of lesions continued for an additional 8 weeks. Complete clearance of AKs was achieved at 12 weeks in 92% (11 of 12) of patients. The total count of AK lesions initially increased more than 2-fold from baseline during weeks 2 and 3 of treatment, as subclinical lesions became evident. By 12 weeks, the total count of lesions had been reduced by more than 99% from baseline. Once-daily 5-fluorouracil 5% cream was well tolerated. On average, patients reported that it was “slightly painful” at week 2 and “not painful at all” at weeks 4 and 8. Once-daily application of 5-fluorouracil 5% cream appears to be an effective and useful alternative to the traditional twice-daily regimen for treatment of AKs.