The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, ey and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with 3H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-ey combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.