Mucositis is a common side effect of cancer chemotherapy for which there is currently no treatment. Irinotecan is a commonly used effective chemotherapeutic agent, causing severe gastrointestinal mucositis and diarrhea. Previous research suggests that palifermin is potentially antimucotoxic. The primary aim of this study was to determine whether palifermin was effective in ameliorating irinotecan-induced gastrointestinal mucositis. We also determined the protective effects of single large and multiple small doses of palifermin. Tumor-bearing DA rats were treated with a single large (10 mg/kg) dose of palifermin 3 days prior to, or multiple small (3 mg/kg day for 3 days) doses of palifermin or vehicle control prior to, receiving 2 doses of 150 mg/kg irinotecan. Animals were killed at 6, 24, 48, 72, 96, 120, or 144 hr after treatment. The primary endpoints were diarrhea and mortality. Gastrointestinal morphometry, histopathology and apoptosis were assessed. Tumor weights and mitoses were measured to ensure palifermin did not promote tumor growth. Data were analyzed using Peritz' F-test, Student's t-test and Tukey-Kramer multiple comparison test. Animals pretreated with palifermin tolerated irinotecan treatment better than control animals with less severe diarrhea (5% in animals receiving 10 mg/kg palifermin, 11% in animals receiving 3 x 3 mg/kg palifermin and 28% in animals receiving irinotecan only) and reduced mortality (2% in animals receiving 10 mg/kg palifermin, 11% in animals receiving 3 x 3 mg/kg palifermin and 28% in animals receiving irinotecan only). Small and large intestinal weights were maintained. Intestinal morphometry was not maintained in palifermin-pretreated rats despite being increased prior to irinotecan treatment. Palifermin pretreatment did not prevent apoptosis that peaked at 6 hr in the jejunum or colon, but prevented apoptosis at 96 hr in the small intestine. Palifermin pretreatment in both treatment regimens significantly reduces diarrhea and mortality following irinotecan administration without adversely affecting tumor growth. This positive response warrants further investigation, particularly in humans.