Heparan sulfate was prepared from the pronase digest of the porcine kidney and modified chemically to yield N-desulfated, N-, O-desulfated, and N- O-desulfated N-acetylated heparan sulfates. Heparan sulfate and its modifications thus obtained were used as substrates to measure the sulfotransferase activity in the microsomal fraction of the endometrium of the rabbit uterus. N-, O-Desulfated heparan sulfate was the best acceptor of sulfate from 3'-phosphoadenylyl [35S] sulfate and intact heparan sulfate was the poorest one. The products of enzymatic reaction were treated with nitrous acid and examined by gel-filtration and electrophoresis. The results indicated that sulfate was almost exclusively incorporated as N-sulfate into all the present substrates.