Chromosomal puffing in the salivary glands of larval Chironomus tentans was developed as a biomarker for genotoxic substances. Reduced chromosomal puffing was considered an indication of decreased RNA synthesis. Third-or fourth-instar larvae were exposed to test chemicals in an artificial substrate under static conditions. Chromosomes from glands of individual larvae were stained with methyl green and pyronin Y. The widths of Balbiani rings I and I1 (large puffs on chromosome IV) were measured. Three carcinogens with different mechanisms of action were tested, benzo[a]pyrene (BaP), actinomycin D (Act D), and dimethylnitrosamine (DMN). Puff size was statistically reduced by all three chemicals with varying potency. Lowest-observable-effect levels (LOEL) were 0.5 nmol BaP, 6.0 nmol Act D, and 243,000 nmol DMN. The degree of response was influenced by exposure time, applied dose, individual sensitivity, and possibly chemical hydrophobicity. Biomarker specificity was determined by testing a weak carcinogen, benzo[e]pyrene (BeP), and an acutely toxic noncarcinogen, naphthalene (NP). The effective dose of BeP (LOEL = 250 nmol) was four orders of magnitude higher than that of BaP. Only lethal doses of NP had statistically significant effects on puff size, LD50 = 25,000 (34,700) nmol. Approximately 40% of the larvae in our laboratory population appeared tolerant to the effects of BaP. Advantages of this biomarker were its association with a known mechanism of action and measurement of the whole-organism integrated response.