Chloroplast ultrastructure, pigment content, and composition were investigated during growth of 'Puma' rye at warm and cold-hardening temperatures. Cytological characteristics of leaf mesophyll cells were altered upon growth at cold-hardening temperatures as indicated by an apparent increase in the amount of cytoplasm. Univacuolate and multivacuolate mesophyll cells were found in leaves from cold-hardened plants, but only univacuolate mesophyll cells were found in leaves from warm-grown plants. Chloroplast ultrastructure was affected by growth at low temperature as indicated by a higher frequency of smaller granal stacks. However, there was no significant difference in the number of grana per chloroplast in mesophyll cells from either warm- or cold-grown plants. Chlorophyll per plastid increased by a factor of 1.67 upon growth at cold-hardening temperatures, as did β-carotene and the xanthophylls. However, there was no significant difference in photosynthetic unit size based on P700 measurements between chloroplasts from warm- or cold-grown tissue. Analysis of freeze-fractured thylakoid membranes indicated a loss in the bimodal nature of particle size distribution on the exoplasmic fracture face and a general increase in particle size on the protoplasmic fracture face upon growth and development at cold temperatures. However, particle densities on the exoplasmic and protoplasmic fracture faces were not significantly different in thylakoids from plastids developed at either warm or cold temperatures. It is concluded that chloroplast ultrastructure and pigment content but not composition are altered upon growth and development at cold-hardening temperatures.