A relatively large number of genes have been described that are required for the normal spatial expression of the genes of the bithorax complex. Most of these regulators appear to act negatively and are required to prevent indiscriminate expression of bithorax complex (BX-C) functions. In this report we examine five negative BX-C regulators to determine whether these are maternally expressed in germ-line derived cells. The genes studied include Additional sex combs (Asx), Polycomblike (Pcl), Sex comb extra (Sce), Sex comb on midleg (Scm), and lethal(4)29 [l(4)29]. The maternal germ-line dependent expression of each of these genes is assessed by comparison of zygotes from mothers whose functional germ cells carry no wild-type alleles to zygotes from mothers whose germ cells contain one wild-type allele. Because mutant alleles of each of the genes studied are recessive lethals, mosaic females with homozygous or hemizygous mutant germ lines were produced by pole cell transplantation. The results demonstrate that all of the negative regulators tested are expressed in the maternal germ line and all play important roles in the regulation of BX-C activities during embryogenesis. The absence of maternally supplied products from all of the genes studied except l(4)29 can be largely or completely compensated for by the activity in the zygote of a paternally contributed wild-type allele. It is argued that, with the exception of l(4)29, the genes studied in this report are qualitatively similar in function to the previously described BX-C regulators Pc, esc, and sxc. The available evidence indicates that genes within this group have functions that are not restricted to the regulation of genes that control segmental identity.