Uniform antibody microdistribution throughout tumor nodules is crucial for antibody-targeted therapy, because non-uniform microdistribution leads to suboptimal therapeutic effect, a commonly observed limitation of therapeutic antibodies. Herein, we evaluated the microdistribution of different doses of intraperitoneally injected fluorescence-labeled full-antibody trastuzumab (15, 50, and 150 microg) and its Fab fragment (trastuzumab-Fab: 15 and 50 microg) in a mouse model of ovarian cancer with peritoneal disseminated tumor. A semiquantitative approach (central/peripheral accumulation ratio; C/P ratio) was developed using in situ fluorescence microscopy. Furthermore, we compared the microdistribution of intact trastuzumab with a mixed injection of trastuzumab and trastuzumab-Fab or serial injections of trastuzumab using in situ multicolor fluorescence microscopy. Fluorescence images after the administration of 15 or 50 microg trastuzumab and 15 microg trastuzumab-Fab demonstrated antibody accumulation in the tumor periphery, whereas administration of 150 microg trastuzumab and 50 microg trastuzumab-Fab showed relatively uniform accumulation throughout the tumor nodule. Using serial injections (19-h interval) of trastuzumab-rhodamine green and carboxytetramethylrhodamine (TAMRA), it was observed that the latterly injected trastuzumab-TAMRA was distributed more centrally than trastuzumab-rhodamine green injected first, whereas no difference was observed in the control mixed-injection group. Moreover, the mixed injection of trastuzumab and trastuzumab-Fab showed that trastuzumab-Fab distributed more centrally than the same amount of co-injected trastuzumab. Our results suggest that the strategies of increasing dose and using Fab fragments can be used to achieve a uniform antibody distribution within peritoneal disseminated nodules after intraperitoneal injection. Furthermore, serial-injection and mixed-injection strategies can modify antibody microdistribution within tumors and have the potential for preferential delivery of anticancer drugs to either the tumor periphery or its center.