A novel biomaterial application of porous microspheres is for sustained delivery of biologically active agents. Recent studies have pointed out the importance of biomaterial porosity in promoting biocompatibility and controlling release rate of active agents. The objective of this research was to investigate the effect of chain-extending agent on the porosity and release behavior of polyurethane (PU) microspheres prepared using a two-step suspension polycondensation method with methylene diphenyl diisocyanate (MDI) as the isocyanate, polyethylene glycol (PEG400) as the diol, and 1,4-butanediol as the chain-extending agent. Chain-extending agent was used to increase the ratio of hard to soft segments of the PU network, and its effect on microsphere morphology was studied with scanning electron microscopy. According to the results, porosity was significantly affected by the amount of chain-extending agent. The pore size decreased as the concentration of chain-extending agent increased from zero to 50 mole%. With further increase of chain-extending agent to 60 and 67%, PU chains became stiffer and formation of pores was inhibited. Therefore, pore morphology was significantly affected by variations in the amount of chain-extending agent. The release behavior of microspheres was investigated with diazinon as the active agent. After an initial burst, corresponding to 3% of the incorporated amount of active agent, the release rate was zero order.