Chloroplasts are ideal hosts for expression of transgenes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Identification of spacer regions to integrate transgenes and endogenous regulatory sequences that support optimal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chloroplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agronomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower [Brassica oleracea], cabbage [Brassica capitata], lettuce [Lactuca sativa], oilseed rape [Brassica napus], petunia [Petunia hybrida], poplar [Populus spp.], potato [Solanum tuberosum], tobacco [Nicotiana tabacum], and tomato [Solanum lycopersicum]) or embryogenesis (carrot [Daucus carota], cotton [Gossypium hirsutum], rice [Oryza sativa], and soybean [Glycine max]), and maternal inheritance of transgenes has been observed. Chloroplast-derived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INFα2b transplastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus [CPV] and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of biopolymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector systems used in these biotechnology applications are described.