We have previously reported the identification of a nonhistone chromosomal protein (nhcp-19; now called HP1) preferentially associated with the heterochromatin of Drosophila melanogaster. A detailed study of the HP1 distribution pattern on polytene chromosomes by immunofluorescent staining, using monoclonal antibody C1A9, has been carried out. The results indicate that this protein is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Analysis of a fourth chromosome insertional translocation T(3;4)f/In(3L)P confirms an autonomous interaction with chromosome 4 material. Similarly, the beta-heterochromatin distal to light on chromosome arm 2L, moved to position 97D2 on chromosome arm 3R in the rearrangement ltx13, is prominently stained using the C1A9 antibody. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent. Thus, the C1A9 antibody may serve as a useful marker for both structural and functional studies of the Drosophila nucleus.