The bacterial isolate GFAJ-1 has been proposed to substitute arsenic for phosphorus to sustain growth. We have shown that GFAJ-1 is able to grow at low phosphate concentrations (1.7 μM), even in the presence of high concentrations of arsenate (40 mM), but lacks the ability to grow in phosphorus-depleted (<0.3 μM), arsenate-containing medium. High-resolution mass spectrometry analyses revealed that phosphorylated central metabolites and phosphorylated nucleic acids predominated. A few arsenylated compounds, including C6 sugar arsenates, were detected in extracts of GFAJ-1, when GFAJ-1 was incubated with arsenate, but further experiments showed they formed abiotically. Inductively coupled plasma mass spectrometry confirmed the presence of phosphorus in nucleic acid extracts, while arsenic could not be detected and was below 1 per mil relative to phosphorus. Taken together, we conclude that GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent, bacterium.