The synthesis of a new series of fluorescent transmembrane probes in which two hydrophilic methyl ester or carboxyl groups are connected by a polymethylene chain, with four, five or six conjugated double bonds in a central position, is reported. The length of the linear structures was designed to match the width of typical lipid bilayers. These bolaamphiphilic compounds result, with overall yields higher than 80%, from an easy PdII‐catalyzed double cross‐coupling between terminal acetylene esters and conjugated 1,ψ‐dihalopolyenes, followed by selective triple bond partial reduction with activated zinc, and iodine isomerization to the all‐(E) isomer. An alternative approach, based on a Stille double cross‐coupling between the appropriate all‐(E)‐ψ‐halopolyenes and (E)‐bis(tributylstannyl)ethene, yielded mixtures that could not be resolved by standard chromatographic methods due to the presence of other simultaneous coupling reactions, which are also discussed in detail. Nevertheless, the Stille method can be of utility for the obtention of carbonyl‐polyene conjugated analogs. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)