Lozenge-shaped poly(4-hydroxybutyrate) single crystals, showing spiral growth, were obtained from a dilute ethanol solution. The crystals gave well-resolved electron diffraction patterns from which the reciprocal lattice parameters a* = 1.305 nm(-1), b* = 2.085 nm(-1) and gamma* = 90 degrees could be determined. Systematic absences occurred at every odd reflection along the two orthogonal axes a* and b*. Therefore, the P(4HB) electron diffraction pattern is consistent with a p2gg two-dimensional symmetry. The unit cell of P(4HB) is orthorhombic with space group P2(1)2(1)2(1) and lattice constants a = 0.775 +/- 0.002 nm, b = 0.477 +/- 0.002 nm and c (fiber axis) = 1.199 +/- 0.002 nm, which is determined by X-ray fiber diagram of a stretched-annealed film combined with electron diffraction patterns of single crystals. There are two chains in one unit cell, which exist in antiparallel arrangement.