Objectives The objective of this study was to evaluate itraconazole 10 mg/ml oral solution for the treatment of Microsporum canis infection using an alternating-week pulse therapy regimen in a controlled laboratory setting. Methods Eighty cats with experimentally induced infections were randomly assigned to treatment (itraconazole vs control [sterile water]), administered 5 mg/kg PO q24h for 1 week on alternate weeks for 5 weeks, followed by a 4 week follow-up period. Topical therapeutic treatment was not administered. Cats were individually housed in stainless steel cages that were cleaned and disinfected daily. Study measures included weekly fungal cultures, clinical lesion scores, Wood's lamp examination and periodic laboratory monitoring. Mycological cure was defined as two consecutive negative cultures. Results Itraconazole-treated cats had significantly greater ( P = 0.0003) mycological cure compared with untreated controls (24/40 [60%] vs 1/40 [2.5%], respectively) and all of these reached clinical cure and had negative final Wood's lamp examinations. Furthermore, 36/40 (90%) treated cats had at least one negative fungal culture by the end of the study vs only 3/40 (7.5%) control cats. For both treatment groups, prevalence of clinical cure peaked at the end of the study (week 9), with 39/40 (97.5%) itraconazole-treated cats and 6/40 (15%) control cats achieving clinical cure. Wood's lamp negative examination rates were significantly greater ( P <0.0001) for itraconazole-treated cats compared with controls (39/40 cats [97.5%] vs 6/40 [15%], respectively) and followed the same pattern of improvement as primary clinical lesions. Conclusions and relevance In this controlled study, orally administered itraconazole using a 5 mg/kg pulse-dose treatment regimen reduced the time to mycological cure and increased both mycological and clinical cure rates compared with untreated controls.