A new allele of one of the metallothionein genes of D. melanogaster, Mtn•3, sheds light on the recent evolution of this gene. In comparison to the previously studied Mtnl allele found in Canton S, this new allele, Mtn•3, produces a transcript that is 49 bases longer and 65–70 % less abundant. We detected Mtn•3 in several laboratory strains as well as in isofemale lines derived from natural populations. Sequence comparison showed that Mtn•3 differs from Mtnl in that it has: (a) base-pair substitution and an extra 49 bp-segment in the 3' untranslated region, (b) a substitution in the coding region that replaces the terminal Glu40 in Mtnl with Lys40, and (c) two base-pair substitutions in the promoter region. The Mtn•3-type was detected in six species of the melanogaster group by restriction analysis, and this result was confirmed by sequencing the D. simulans Mtn gene. Thus Mtn•3, which produces a less abundant transcript, appears to be the oldest of the two alleles. We also found that the duplications previously isolated from natural populations all derived from Mtnl, the more recent allele. Thus, two evolutionary steps: Mtn•3 to Mtnl and Mtnl to Dp(Mtnl), are accompanied by an overall 5- to 6-fold increase of RNA accumulation. The two changes seem to have occurred in non-African populations since Mtn•3 but not Mtnl was detected in our sample from tropical Africa, while Mtnl and Dp (Mtnl) are prevalent in European and North American samples.