An experiment was conducted to determine the types, extent, and heritability of new phenotypic variants recovered from carrot cell cultures initiated from mature tap-root explants of the male-fertile carrot (Daucus carota L.) `Slendero'. Embryogenic callus was transferred to plant-growth-regulator-free medium 66 days after culture initiation, and regenerated plantlets were harvested and eventually planted in a field. The tap roots of mature regenerated plants were vernalized at 5C for 9 weeks and replanted. Of 31 flowering regenerants, 25 exhibited some form of petaloid male sterility; the remaining six regenerants were male fertile. All plants from the same original explant were either all sterile or all fertile. Three generations of sterile regenerant × petaloid cytoplasmic male sterility (CMS) maintainer (M) progeny tests showed that the new CMS behaved in a similar manner to that previously reported. Comparison of mitochondrial DNA restriction patterns of sterile and fertile regenerants with those of `Slendero', petaloid CMS, petaloid M, and brown anther CMS lines resulted in the following conclusions: 1) the sterile regenerants exhibited patterns identical to the known petaloid CMS and 2) the fertile regenerants were different from the original `Slendero' and the sterile regenerants and nearly identical to a known petaloid CMS M line. The high frequency of CMS among regenerants from `Slendero' carrot cell cultures may provide an efficient method to develop sterile M tandem lines and corresponding new hybrid varieties.