To better understand and design microsphere systems for the locoregional delivery of anticancer drug combinations to solid tumors, (1) the cytotoxicity of microsphere-delivered mitomycin C (MMC) was evaluated and (2) various schedules of MMC and doxorubicin (Dox) were tested for their toxicity in vitro towards a murine breast cancer cell-line, EMT6. To accomplish the former MMC was loaded onto oxidized sulfopropyl dextran microspheres, released in a pH 7.4 buffer solution and tested for its potency against EMT6 cells versus a standard MMC solution. For the latter EMT6 cells were exposed to MMC or Dox as single agents or together using various drug concentrations and schedules. The efficacy of the treatments was measured using a clonogenic assay. MMC released from the microspheres showed similar activity against EMT6 cells to freshly prepared MMC solutions. Greater-than-additive toxicity was observed when MMC was given either simultaneously or after Dox exposure. In contrast, administration of MMC before Dox exposure resulted in toxicity that ranged from additive to sub-additive; this reduced toxicity was mainly due to increasing cell density arising from the design of the assay. These results help explain our previous in vivo investigations using microsphere-delivered combinations of the same agents in EMT6 solid tumors.