Objectives Evaluate development of macaque embryos in simple chemically-defined, protein-free culture medium, and determine if a 2-step progressive culture system can enhance blastocyst development and zona escape. ABSTRACT:In experiment 1, in vitro fertilized (IVF) pronucleate stage embryos (n = 9 monkeys) were cultured in each of three treatments 1) HECM-6 (chemically-defined, protein-free medium), 2) CMRL-BCS (modified CMRL-1066 medium containing 20% bovine calf serum; BCS), 3) 2-step culture procedure (HECM-6 through 8-12-cell stage/CMRL-BCS beyond 8-12-cell stage). Optimal development was attained equally (p r 0.05) with embryos cultured in CMRL-BCS or the 2-step procedure (48% and 61% blastocysts, respectively). HECM-6 alone supported development to the morula stage (72%) equally well as CMRL-BCS (80%) or the 2-step procedure (69%), but not to the blastocyst stage (22% vs. 48% and 61%, respectively). Hatching of blastocysts was essentially limited to serum-containing media (CMRL-BCS 31%; 2-step procedure 44%). In experiment 2, IVF embryos (n = 9 monkeys) were placed into each of four 2-step treatments 1) HECM-6 through 8-12-cell stage/CMRL-BCS beyond 8-12-cell stage, 2) HECM-6 through 8-12-cell stage/HECM-6-BCS beyond 8-12-cell stage, 3) HECM-6 through morula stage/CMRL-BCS beyond morula stage, 4) HECM-6 through morula stage/HECM-6-BCS beyond morula stage. Greater (p s 0.05) percentages of embryos developed into blastocysts, expanded blastocysts and hatched blastocysts when switched at the 8-12-cell vs. morula stage into second step medium. When transferred into BCS-containing medium at either the 8-12-cell or morula stage, embryos underwent blastulation and expansion equally well in CMRL-BCS vs. HECM-6-BCS. This work is the first to produce IVF primate blastocysts in chemically-defined, protein-free medium. While primate embryos can form morulae in such a medium, requirements for blastocoel formation appear more demanding, and may be acquired as early as the 8-cell stage. Keywords rhesus monkey, pronucleate stage embryo, zona escape, HECM-6 medium