In October 2003, the NIH established three extramural "Exploratory Centers for Human Embryonic Stem Cell Research." Our center acquired 15 of the 22 NIH-approved cell lines. Lines were tested for: (a) freedom from mycoplasma contamination; (b) appropriate pattern of gene expression during self-renewal and differentiation; (c) ability to adapt to uniform culture conditions; (d) ability to grow at clonal densities; (e) karyotype; (f) growth efficiency; and (g) efficiency of stable transfection following electroporation. One line harbored mycoplasma. Ten lines were converted to uniform conditions. Nine lines were fully characterized. Human ESC (hESC) lines varied markedly with respect to growth efficiency as measured by the amount of time it took to plate and double (31-57 hours), cloning efficiency (0.8%-9.2%), and stable transfection rates following electroporation (0%-53% relative to a standard mouse ESC line). One hESC line had an unstable karyotype at an early passage. Modifications of the proposed Material Transfer Agreements with hESC suppliers were required to improve accessibility to hESC lines by local researchers. The NIH-approved hESC lines vary in their behavior in culture. Many hESC lines can be maintained using culture conditions less onerous than those recommended by their suppliers. Intellectual property issues pose a significant obstacle to research using NIH-approved hESC lines.