AbstractRetinoic acid (RA) is purported to be required for expression of genes controlling proximodistal (Meis2) or anteroposterior (Shh) limb patterning. Embryos lacking RDH10, the primary enzyme synthesizing retinaldehyde during mouse development, survive until E14.5 with stunted forelimbs but apparently normal hindlimbs. Using embryos carrying the RARE‐lacZ RA‐reporter transgene, we show that endogenous RA activity in Rdh10trex/trex mutants is detected in neuroectoderm but not limbs during initiation and patterning. Treatment of Rdh10 mutants with 25 nM RA restores RARE‐lacZ activity to limb mesoderm, validating RARE‐lacZ and verifying that RA is absent in mutant limbs. In Rdh10 mutants, hindlimbs exhibit normal Meis2/Shh expression and skeletal patterning, and although forelimbs are growth‐retarded their Meis2 expression remains normal. Later in development, Rdh10 mutants lack interdigital RA activity and accordingly fail to exhibit normal loss of interdigital mesenchyme. These findings demonstrate that RA is unnecessary for limb patterning but required later for interdigital tissue loss. Developmental Dynamics 240:1142–1150, 2011. © 2011 Wiley‐Liss, Inc.