The formation and maintenance of the highly regular organization of membrane systems and proteins in striated muscle require specific membrane-membrane and membrane-cytoskeleton interactions. The development of T-tubules and sarcoplasmic reticulum (SR) was followed in gastrocnemius muscle fibers from chicken embryos between 12 days (E12) and 21 days (E21) of incubation, with particular attention to their relationship with one another and with the myofibrils. The fluorescent lipid analog DiIC16[3] was used to label either the external membranes (plasmalemma and transverse (T)-tubules) or the internal SR in living and fixed muscle. Short membrane invaginations can first be seen in fibers at E14, and at E15 longitudinal T-tubules appear in the periphery of the fibers. A complex network of T-tubules filling the whole fiber diameter develops suddenly at E16. In contrast, SR is abundant at the earliest observed stage (E12) and forms regularly spaced cross striations located at the I-Z-I bands. These correspond to a specific accumulation of smooth membranes around the Z-discs seen in electron micrographs. While SR is specifically associated with the newly formed myofibrils in the periphery of the fibers, the disposition of early T-tubules shows little specific relationship to either SR or the myofibrils. However, electron microscopy shows that junctions between T-tubules and SR are formed during this period (Takekura and Franzini-Armstrong, submitted for publication). Junctions do not acquire a specific relation to the myofibrils until around hatching when triads begin to reorganize into their mature location, the A-I junction. These findings indicate three key events in the organization of T-tubules and SR in the sarcomeres: (1) early SR/Z-line interactions independent of T-tubules; (2) SR/T-tubule interactions to form the triad junctions, independent from the myofibrils; and (3) the late association of the junctional complexes with the myofibrils at the A-I border.