O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.