Cell-free 100,000 g supernatants from human lung converted PGD2 into a product which on HPLC was indistinguishable from 9 alpha, 11 beta-PGF2. The rate of reaction was relatively slow (0.30 +/- 0.03 pmol/min/mg protein). In the presence of NAD+, 9 alpha, 11 beta-PGF2 was itself metabolized at a rate of 1.46 +/- 0.3 pmol/min/mg protein. The product of this reaction was less polar than the substrate and eluted with an HPLC retention time similar to that seen in our previous study where it was identified by GC/MS as being 15-keto-9 alpha, 11 beta-PGF2. There was no evidence for the formation of 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. The sequence of metabolism of PGD2 was further established in cell-free supernatants prepared from guinea-pig liver and kidney which have previously been shown to be rich in both 11-ketoreductase and 15-hydroxy-prostaglandin dehydrogenase (PGDH) activities. Reactions contained both NAD+ and a NADPH-regenerating system and demonstrated the sequential metabolism of PGD2 to 9 alpha, 11 beta-PGF2 and ultimately 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. Under these conditions the rate of the C-15 oxidation reaction was slower than that of 11-ketoreduction. These observations provide further support for our previous suggestions that the 11-ketoreduction of PGD2 is followed by a PGDH-type reaction, and that these reactions are likely to occur sequentially in vivo provided that the appropriate cofactors are present.