Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle‐specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0–24 h postmitosis) by video time‐lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x‐h‐old to define their age relative to their mitotic “birthdate.” All 6 h myoblasts that emerged in a mitogen‐rich medium were desmin+ but only 50% were positive for a α‐actin, troponin‐I, α‐actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up‐regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were α‐actinin+ or zeugmatin+ but MyHC− or titin− whereas others were troponin‐I+ or MyHC+ but α‐actinin− or α‐actin−. In 15‐h‐old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle‐shaped cells; and (2) the sarcolemma became fusion‐competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP‐C (C protein). The emergence of one, or a string of ∼ 2 μ long sarcomeres, was invariably characterized by the localization of myomesin and MyBP‐C to their mature positions in the developing A‐bands. The latter group of A‐band proteins may be rate‐limiting in the assembly program. The great majority of rnyoblasts stained positively for desmin and rnyofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle‐specific proteins in vitro fully recapitulates myofibrillar assembly steps in rnyoblasts of the myotome and limb bud in vivo, as well as in nonrnuscle cells converted to myoblasts by MyoD. We suggest that this cell‐autonomous myoblast differentiation program may be blocked at different control points in immortalized rnyogenic cell lines. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc.