The structure of the mRNA for apolipoprotein II (apo-II), a major avian estrogen-responsive yolk protein, has been investigated. Primer extension using a cDNA probe-primer revealed six forms of mature apo-II mRNA. S1 and primer extension analysis with intron probes showed that only three of these forms arise due to multiple sites of transcription initiation on the apo-II gene. To investigate the basis for the additional forms of apo-II mRNA, we examined their nucleotide sequence. The sequence obtained between -10 and -42, relative to translation initiation, is identical to that reported by Wieringa et al. (Wieringa, B., AB, G., and Gruber, M. (1981) Nucleic Acids Res. 9, 469-499). At position -43, however, sequence heterogeneity appears. The minor form of the sequence starting at -43 and extending in a 5' direction corresponds exactly to the published mRNA sequence. The major form of the sequence has the insertion, 5'-CAG-3', at this position. The apparent basis for this heterogeneity is an unusual intron-exon border which violates the consensus sequence found in comparable positions in most eukaryotic genes by the existence of two adjacent 5'-CAG-3' triplets after the pyrimidine (Y)-rich track. The processing ratio between intron removal at the upstream and downstream AG dinucleotide is approximately 2.5:1. This result demonstrates that the splicing mechanism allows spatial flexibility in the positioning of the highly conserved YAG triplet within the consensus sequence at the 3' splice site.