The puffing pattern of polytene chromosomes in salivary glands from Chironomus pallidivittatus larvae and prepupae has been studied in glutaraldehyde-acetic acid fixed, lactic acid flattened preparations. Some observations were also made on F1 hybrid species C. pallidivittatus X C. tentans. Concerning the situation of puffing in Balbiani rings (BR), 2.783 chromosomes IV from 188 animals were scored. In standard 4th instar larvae, BR2 appears expanded, BR3 smaller but not collapsed and BRI either reduced of collapsed. During the first days following the red-head stage, which signals the beginning of the 4th instar, larvae show a large BR1; later it reduces and tends to collapse. At the end of the 4th instar, prepupae again may present an expanded BR1. On the contrary, the size of BR2 and BR3 remains unchanged from the red-head stage to the prepupa. A variable accumulation of droplets has been observed to occur in BR2 and BR1 from dated larvae and prepupae.--A characteristic pattern of puffing was found in prepupae, which consisted in the appearance of conspicuous puffs at regions I-6B, I-7B, I-7B, I-18C, III-9B and IV-4B. Puffs at I-2B, I-3B, I-9A,I-11C,II-4A, and IV-4B were observed during most of the 4th larval instar, as well as in late larvae and prepupae. Among all these puffs, those at I-7B, I-9A, I-17B, and IV-4B frequently showed variable amounts of droplets.