Composition of seed storage proteins in in vitro cultured cotyledons is known to be affected by exogenous application of abscisic acid (ABA) and sulfur deficiency. In this paper, we analyzed effects of exogenous ABA on regulation of soybean seed storage protein accumulation in relation to that of sulfur deficiency. When exogenous ABA was applied at 10 μM and 100 μM, accumulation of the β subunit of β-conglycinin, a major seed storage protein, increased, whereas that of glycinin, another major seed storage protein, decreased. Free sulfate concentrations in cotyledons were not significantly affected by application of 10 μM ABA, but doubled by application of 100 μM ABA. As we have previously shown that concentrations of O-acetyl-L-serine (OAS), a precursor of cysteine biosynthesis, in in vitro cultured soybean cotyledons increased under sulfur deficiency, effects of exogenous ABA application on OAS concentration in soybean cotyledons were determined. The concentrations of OAS increased in accordance with the increase of exogenous ABA concentrations. These results suggested that exogenous application of ABA induces changes in soybean storage protein composition through the increase of OAS concentration.