Retinoic acid (RA) has dramatic effects on the pattern of developing and regenerating vertebrate limbs. These effects are considered to result from RA-induced changes in the positional identity of limb cells, and involve the formation of extra structures. Whether the growth required to form the supernumerary parts of the pattern is a primary effect of RA treatment or a secondary effect that follows after a change in positional identity is not at present known. In this paper we have investigated the effects of RA treatment on the growth of cells from anterior and posterior halves of mouse limb buds in vitro. We observed that under our culture conditions, limb bud cells treated with 1 nM to 1 μM RA (0.3 ng/ml to 300 ng/ml) continue to grow but do so at a significantly slower rate than control cultures. There is a maximum inhibition of growth (50% of controls) between 10 nM and 100 nM RA, which corresponds to the measured range of concentrations of RA in vivo. Our observation of a significant decrease in growth rate over a wide range of RA concentrations is consistent with comparable reports of growth inhibition for a large number of other cell types in vitro as well as with the observation that exogenous RA inhibits blastemal growth in amphibians during the period of exposure to RA. We propose that the effects of RA on growth, either enhancement in vivo or reduction in vitro, can be seen as consequences of the ability of RA to alter positional identity. Hence, when RA is administered to limb cells in vitro, it is likely that the non-posterior cells in both anterior and posterior cultures are converted to posterior identity. The resulting decrease in growth can be interpreted as a consequence of the increased uniformity of positional information in vitro. © 1992 Wiley-Liss, Inc.