We have used low-level photocrosslinkage to study chromatin effects on psoralen intercalation at specific DNA sequences of various complexities in intact, cultured, Drosophila cells. Alkali-denatured DNA connected in both strands to a 4,5',8-trimethylpsoralen (TMP) interstrand crosslink is insensitive to digestion by the single strand-specific nuclease S1 and does not hybridize to complementary DNA. Crosslink number at any ultraviolet light exposure increases in proportion to the concentration [PS] of TMP dark binding sites that are occupied. The crosslinking constant, K, is the increase in crosslink number per length DNA per increment [PS]. Many factors influence K, including sequence composition and ionic strength. We show here that the ratio of K at any specific sequence (Kh, from hybridization measurements) to Kh at any other specific sequence or to K of total DNA (Kf, from fluorimetry measurements) can be calculated from measurements of crosslinkage, the mass fraction of the sequence in question or of total DNA that is connected in both strands to a crosslink. When crosslinked and uncrosslinked DNAs fragmented by mechanical shear were mixed in known proportions, Kf exceeded Kh of a single-copy gene by 15%. We treated cells with TMP plus near ultraviolet light, then tested for crosslinkage and for hybridization. A single-copy, larval gene at 70D, and a 250-copy type 1 ribosomal DNA intervening sequence, neither of which is transcribed in these cells, were as sensitive to crosslinkage as total, cell DNA. However, single-copy, heat shock gene sequences from loci 63BC and 95D, and the 180-copy ribosomal DNA coding sequence were more sensitive to crosslinkage than total DNA in the same preparations. The excess was largest in the shortest fragments, indicating a localized effect. The same sequences were crosslinked less readily than total DNA in vitro; we calculate a 3.4 to 3.8-fold excess crosslink number in these sequences due to chromatin microenvironment. We tested for effect of transcriptional induction on crosslink sensitivity in the heat shock genes. At low [TMP], heat shock stimulated crosslinkage at or very near heat shock genes in cells, but not in other sequences or in naked DNA. However, overall crosslink sensitivity was unaffected by heat shock. This suggests that transcription increased the affinity of some heat shock gene DNA binding sites for TMP without increasing the number of such sites.