Forelimbs of Day 11 and Day 12 embryonic mice were excised and cultured for 3 d in the presence of either 0.25 μg (8×10-7 M), 0.5 μg(1.7×10-6 M), or 1.0 μg (3.3×10-6 M) of all-rans retinoic acid (RA) per milliliter of culture medium. Cultured limbs were fixed, stained, and mounted whole on glass slides and evaluated with computerized optical image analysis for RA-induced effects on the area and shape of the total limb and individual bone anlagen. Relative effects of RA on total bone, soft tissue, long bone, and paw regions were also examined. With Day 11 forelimbs total bone area was increased by 10.5% by the low dose of RA. The increase was mostly in long bones and at the expense of soft tissue. Total bone area was increased 9.3% with Day 12 forelimbs. This increase was primarily in the paw. The high dose of RA decreased Day 11 forelimb area, primarily affecting long bones. Day 12 forelimbs were not significantly affected by the high dose of RA. Effects of the imtermediate dose were primarily limited to reduction in soft tissue area. Long bone:paw and soft tissue: bone ratios reflected these effects. The high dose produced a consistent rounding or shortening of Day 11 forelimb bones. On Day 12 0.5 μg/ml RA produced an inconsistent pattern of rounding of bone anlagen. Treatment with the high dose on Day 12 produced angular rather than rounded contours in many cases, as indicated by shape factor values closer to zero than obtained with controls. These data show that direct exposure to RA can affect both the size and shape of bone anlagen of the developing limb; the low dose enhances and the high dose depresses development. The results support previous studies which suggest that RA may play a critical role in the control of cell activities such as cell migration, proliferation, and cytodifferentiation in the development of the cartilaginous bone anlagen.