Streptolysin O (SLO) is a known bacterial protein that forms very large pores in the plasma membranes of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effects of SLO treatment in donor cells that generate pores in the plasma membrane during in vitro development of porcine reconstructed embryos. In experiment 1, fetal fibroblast cells were trypsinized, treated with SLO for 0, 30, 50 and 70 min and then their fusion rates and developmental capabilities were tested following reconstruction. SLO treatment for 50 min produced a higher blastocyst formation rate compared with the other treatments. In experiment 2, fetal fibroblasts were treated with 200 ng/ml SLO for 50 min at the confluent or single-cell stages of the nuclear transfer protocol (i.e., before and after trypsinization, respectively), and the in vitro development of the resulting porcine reconstructed embryos was investigated over 6 days in culture. Oocytes receiving cells treated with SLO post-trypsinization showed higher fusion and blastocyst formation rates compared with those receiving untreated cells. Collectively, these findings show that SLO-mediated permeabilization of porcine fetal fibroblast cells appears to improve the fusion rates and in vitro development of porcine reconstructed embryos.