A second chloroplast acyl carrier protein (denoted ACPII) has been purified to homogeneity from greening barley seedlings. Both the previously characterized ACP I and ACP II are active in fatty acid synthesis but differ significantly in their amino acid composition. Comparison of the amino-terminal sequences of the two proteins reveals striking differences as indicated by the underlines. ACP II: NH2-AKKETVEKV?DIVKSQLALSDDDE-, ACP I: NH2-AAMGEAOAKKETVDKV(C?)MIVKKQLAVPDGTP-. We thus deduce that the two ACPs are coded for by different genes. Both ACPs are present in dark and light grown seedlings although in sligthly different proportions. The possible functional significance of two or more ACPs within the chloroplast is discussed in relation to plant fatty acid and glycerolipid biosynthesis.