Liposomes can selectively target cancer sites and carry payloads, thereby improving diagnostic and therapeutic effectiveness as well as reducing toxicity. To evaluate therapeutic strategies, it is essential to use animal models reflecting important safety aspects before clinical application. As our previous study found that a high dosage (185 of MBq) of 188Re-N,N-bis (2-mercaptoethyl)-N’,N’-diethylethylenediamine-labeled pegylated liposomes (188Re-liposome) induced a decrease in white blood cell (WBC) count in Sprague–Dawley rats 7 days postinjection, the objective of the present study was to investigate extended acute radiotoxicity of 188Re-liposome. Rats were administered via intravenous (i.v.) injection with 188Re-liposome (185, 55.5 and 18.5 MBq), normal saline as a blank control or non-radioactive liposome as a vehicle control. Mortality, clinical signs, food consumption, body weights, urinary, biochemical and hematological analyzes were examined. In addition, gross necropsy and histopathological examinations were also performed at the end of the follow-up period. None of the rats died and no clinical sign was observed during the 28-day study period. Only male rats receiving 188Re-liposome at a high dosage (185 MBq) displayed a slight weight loss compared with the control rats. In both male and female rats, the WBC counts of both high-dose and medium-dose (55.5 MBq) groups reduced significantly 7 days postinjection, but recovered to the normal range on Study Day 29. There was no significant difference in urinary analyzes, biochemical parameters and histopathological assessments between the 188Re-liposome-treated and control groups. The information generated from the present study on extended acute toxicity of 188Re-liposome will serve as a safety reference for radiopharmaceuticals in early-phase clinical trials. Copyright © 2012 John Wiley & Sons, Ltd.